TRIBE

TRIBE

TRIBE identifies the target RNA sequences of RBPs in vivo by modifying the RNA sequence using fusion proteins. The fusion protein consists of the RBP of interest, which binds to the target RNA, and the catalytic domain of adenosine deaminase acting on RNA (ADAR), which irreversibly modifies the proximal adenosine to inosine to serve as a marker during sequence analysis.

First, fusion proteins with the RBP of interest are cloned into the animal model and expressed along with fluorescent marker proteins. After activating the expression of the fusion proteins, mRNA from the cells of interest are isolated using oligo(dT) beads. mRNAs are reverse-transcribed using poly(dT)-T7 primers and random-T7 primers to reduce 3' bias. The nascent cDNA strands are input into the TruSeq RNA Library Prep Kit to generate the cDNA library for sequencing.

Pros:

• In vivo identification of RNA targets of RBPs in specific cell types
• Not restricted to the specificity of antibodies
• Can be performed in a small number of specific cells

Cons:

• Requires assembly of fusion proteins
• Requires an adenosine proximal to the RBP binding site
• Catalytic domain of ADAR has a strong preference for double-stranded RNA

• TRIBE: McMahon A. C., Rahman R., Jin H., Shen J. L., Fieldsend A., et al. (2016) TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins. Cell 165: 742-753

1. McMahon A. C., Rahman R., Jin H., et al. TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins. Cell. 2016;165:742-753