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

Abstract

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 provides 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. GRAPHICAL ABSTARCT O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/631239v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@14e97d8org.highwire.dtl.DTLVardef@d30038org.highwire.dtl.DTLVardef@b39459org.highwire.dtl.DTLVardef@1d0679f_HPS_FORMAT_FIGEXP M_FIG C_FIG