High-throughput mapping of modular regulatory domains in human RNA-binding proteins

RNA regulation is central to tuning gene expression and is controlled by thousands of RNA-binding proteins (RBPs). While many RBPs require their full sequence to function, some act through modular domains that recruit larger regulatory complexes. Mapping these RNA-regulatory effector domains is important for understanding RBP function and designing compact RNA regulators. We developed a high-throughput recruitment assay (HT-RNA-Recruit) to identify RNA-downregulatory effector domains within human RBPs. By recruiting over 30,000 protein tiles from 367 RBPs to a reporter mRNA, we discovered over 100 RNA-downregulatory effector domains in 86 RBPs. Certain domains-for instance, KRABs-suppress gene expression upon recruitment to both DNA and RNA. We engineered inducible synthetic RNA regulators based on NANOS that can downregulate endogenous RNAs or maintain reporter expression at defined intermediate levels, as predicted by mathematical modeling. This work serves as a resource for understanding RNA regulators and expands the repertoire of RNA control tools. A record of this paper's transparent peer review process is included in the supplemental information.