Fast and robust characterization of RNA targets of RNA-binding proteins by RNA-editing strategy coupled with machine learning in human embryonic stem cells

RNA-binding proteins (RBPs) paly critical roles in regulating the maintenance and differentiation of embryonic stem cells. A critical step in understanding the functions of RBPs is the identification of their bound RNA transcripts. Towards this aim, we developed an inducible strategy named as RNA-Editing-Based-RNA-seq (REB-seq) to capture the potential RNA targets bound by the RBPs-of-interest. Fused with the catalytic domain of ADAR or APOBEC1 that mediate the A to I (A to G) or C to U (C to T) editing, respectively, REB-seq identifies the bound RNAs by transcriptome profiling without the necessity of high-performance antibodies for the RBPs. Using REB-seq, we characterized the mRNAs bound by the m6A readers IGF2BP1, IGF2BP2, and IGF2BP3 in human embryonic stem cells. We also incorporated machine learning strategy to the analysis of REB-seq data to improve the accuracy of target RNA characterization. Furthermore, REB-seq identifies associated single nucleotide polymorphisms that may affect the RBP binding and imply the disease pathogenesis. Collectively, REB-seq coupled with machine learning can be applied to capture RNA transcripts bound by RBPs-of-interest easily and robustly. hESCs were cultured with Nuwacell® ncTarget hPSC Medium on Matrigel-coated plates. Control hESCs, hESCs with tetON-overexpression of ADAR, hAPOBEC, rAPOBEC, ADAR-IGF2BP1, hAPOBEC-IGF2BP1, rAPOBEC-IGF2BP1, ADAR-IGF2BP2, hAPOBEC-IGF2BP2, rAPOBEC-IGF2BP2, ADAR-IGF2BP3, hAPOBEC-IGF2BP3, rAPOBEC-IGF2BP3 were induced with doxcycycline for 24 hours before extracting total RNA for RNA-seq.