Comparison of TRIBE and STAMP for identifying targets of RNA binding proteins in human andDrosophilacells

Abstract

RNA binding proteins (RBPs) perform a myriad of functions and are implicated in numerous neurological diseases. To identify the targets of RBPs in small numbers of cells, we developed TRIBE, in which the catalytic domain of the RNA editing enzyme ADAR (ADARcd) is fused to an RBP. When the RBP binds to an mRNA, ADAR catalyzes A to G modifications in the target mRNA that can be easily identified in standard RNA sequencing. In STAMP, the concept is the same except the ADARcd is replaced by the RNA editing enzyme APOBEC. Here we compared TRIBE and STAMP side-by-side in human andDrosophilacells. The goal is to learn the pros and cons of each method so that researchers can choose the method best suited to their RBP and system. In human cells, TRIBE and STAMP were performed using the RBP TDP-43. Although they both identified TDP-43 target mRNAs, combining the two methods more successfully identified high-confidence targets. InDrosophilacells, RBP–APOBEC fusions generated only low numbers of editing sites, comparable to the level of control editing. This was true for two different RBPs, Hrp48 and Thor (DrosophilaEIF4E-BP), indicating that STAMP does not work well inDrosophila.