RBPscan: A Quantitative, In Vivo Tool for Profiling RNA-Binding Protein Interactions

RNA-binding proteins (RBPs) are essential regulators of gene expression at post-transcriptional level, yet obtaining quantitative insights into RBP–RNA interactions in vivo remains a challenge. Here we developed RBPscan, a method that integrates RNA editing with massively parallel reporter assays (MPRAs) to profile RBP binding in vivo. RBPscan fuses the catalytic domain of ADAR to the RBP of interest, using RNA editing of a recorder mRNA as a readout of binding events. We demonstrate its utility in zebrafish embryos, human cells, and yeast, where it quantifies binding strength, resolves dissociation constants, identifies high-specificity motifs for a variety of RBPs, and links binding affinities to their impact on mRNA stability. RBPscan also provide positional information of conserved and novel Pumilio-binding sites in lncRNA NORAD. With its simplicity, scalability, and compatibility across systems, RBPscan offers a versatile tool for investigating RBP–RNA interactions and complements established methods for studying post-transcriptional regulatory networks. Overall design: RBPscan analysis of hsaPum2 binding to various PREs was performed in HEK293T cells. Expression of the hsaPum2-ADAR fusion protein was induced by doxycycline. The reporter mRNA library contained 1,000 distinct PREs and other control sequences. Each experiment was conducted in three biological replicates.