Directed protein evolution of an RNA editor for single molecule RNA-binding protein co-occupancy (II)
RNA base editors have become powerful tools for both therapeutic and research purposes. However, currently available RNA editors such as ADAR or APOBEC family proteins have limitations due to RNA structure and sequence dependence. Here, we designed a protein engineering platform for putative RNA editor screening and directed enzyme evolution. We engineered an RNA A-to-I base editor (rABE) that has high activity, low local sequence or structure bias, and low background. Utilizing rABE, we present REMORA (RNA Encoded Molecular Recording in Adenosines), a new strategy to measure RNA-binding events on single RNA molecules in cells. In REMORA, adenosine deamination serves as a molecular record of RNA-protein interactions that are identified by mutations by sequencing. By combining our improved A-to-I RNA deaminase with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, our approach enables simultaneous recording of the locations two RNA binding proteins on single mRNA molecules. Orthogonal RNA molecular recording of two Pumilio family proteins, PUM1 and PUM2, reveals that PUM1 competes with PUM2 for some but not all Pumilio binding sites in cells, despite having the same in vitro binding preferences. Our work thus measures competition between RNA-binding proteins for RNA sites in cells, and our genetically encodable RNA deaminase enables single-molecule identification of RNA-protein interactions with cell type specificity. Overall design: Stable HEK293T cell lines expressing RBP-rABE, RBP-APOBEC1 or the deaminase only controls were generated with lentivral infection. Expression was driven by doxycycline promoters and cells were treated with 1000 ng/ml doxycycline for 48 hours.