Abstract

Protein-RNA interactions govern nearly every aspect of RNA metabolism and are frequently dysregulated in disease. While individual protein residues and RNA nucleotides critical for these interactions have been characterized, scalable methods that jointly map protein- and RNA-level determinants remain limited. RNA deaminase fusions have emerged as a powerful strategy to identify transcriptome-wide targets of RNA-binding proteins by converting binding events into site-specific nucleotide edits. Here, we demonstrate that this 'RNA recording' approach enables high-throughput mutational scanning of protein-RNA interfaces. Using the λN-boxB system as a model, we show that editing by a fused TadA adenosine deaminase directly correlates with binding affinity between protein and RNA variants in vitro. Systematic variation of RNA sequence context reveals a strong bias for editing at UA dinucleotides by the engineered TadA8.20, mirroring wild-type TadA preferences. We further demonstrate that stepwise recruitment of the deaminase using nanobody and protein A/G fusions maintains both sequence and binding specificity. Stable expression of the TadA fusion in human cells reproduces in vitro editing patterns across a library of RNA variants. Finally, comprehensive single amino acid mutagenesis of λN in human cells reveals critical residues mediating RNA binding. Together, our results establish RNA recording as a versatile and scalable tool for dissecting protein-RNA interactions at nucleotide and residue resolution, both in vitro and in cells.