SRP437687 PRJNA972653 GSE232518 Expanded repertoire of RNA-editing-based detection for RNA binding protein interactions (4) RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To expand the collection of rBEs suitable for editing-based RBP-RNA interactome studies, we developed experimental and computational assays to systematically evaluate the editing activities of over thirty A-to-I and C-to-U rBEs in human cells. We identify rBEs that outperform Drosophila ADAR (TRIBE) and mammalian APOBEC1 (STAMP) in the characterization of the binding patterns of sequence-specific and broad-binding RBPs and recommend rBEs that pair effectively in dual-RBP-based applications. We demonstrate that the optimal choice of single or multiple rBEs to fuse to a given or pair of RBPs depends on the editing biases of the rBEs and the binding preferences of the RBPs. Our framework ENGRAVe (Editing nucleotides with general RNA-base-editor variants) frames the use of and expands the choices of rBEs for the next generation of RBP-RNA target discoveries. Overall design: Plasmids encoding RPS2-rBE fusions were transiently transfected into HEK293XT cells. Then, 24 hrs after transfection, construct expression was induced by the addition of doxycycline (Dox) at 2 µg/mL for 24 hrs. After, Torin-1 (100 nM final concentration in the wells) dissolved in DMSO or DMSO without Torin-1 (vehicle) was added, and the cells were incubated for 48 hrs before extraction of DNA-free total RNA and sequencing library preparation. GSE232518 pubmed 38287010 pubmed 39152130 parent_bioproject PRJNA972650