PIE-Seq: Identifying RNA-binding Protein Targets by Dual RNA-deaminase Editing and Sequencing

RNA-binding proteins (RBPs) are essential for posttranscriptional gene regulation, but it remains a challenge to identify RNA targets of RBPs from specific cell types. Here we present PIE-Seq to investigate Protein-RNA Interaction with dual-deaminase Editing and Sequencing by conjugating C-to-U and A-to-I RNA deaminases to RBPs. We benchmark the method and demonstrate its sensitivity in single cells, its application in the developing brain, and its scalability with 25 human RBPs involved in diverse biological functions. Bulk PIE-Seq reveals canonical binding features and target genes for RBPs such as PUM2 and NOVA1, and nominates new target genes for most tested RBPs such as SRSF1 and TDP-43/TARDBP. Integrative analyses show that homologous RBPs frequently bind to comparable sequences and target gene sets while different RBP families bind to distinct targets, indicating that PIE-Seq is robust to uncovering RBP targets. Single-cell PIE-PUM2 uncovers comparable targets to bulk samples and applying PIE-PUM2 to the developing mouse neocortex identifies neural-progenitor- and neuron-specific target genes. In summary, the dual-deaminase PIE-Seq joins the editable nucleotide space for single editors and provides an orthogonal approach and resource to uncover RBP targets in mice and human cells. HEK293FT cells or developing dorsal brain cells were transfected with different plasmids for 48 or 24 hours, transfected cells were sorted by FACS, and then total RNA was extracted from each group for RNA-Sequencing.