High-throughput discovery of regulatory domains in RNA-binding proteins

RNA plays an integral role in tuning gene expression and is regulated by thousands of RNA-binding proteins (RBPs). We develop and use a high-throughput recruitment assay (HT-RNA-Recruit) to identify regulatory domains within human RBPs by testing 30,000 protein tiles from 367 human RBPs for regulatory capacity when recruited at a reporter mRNA. We discover over 100 unique regulatory domains in 86 distinct RBPs, presenting evidence that RBPs are modular, containing functionally separable domains that dictate their post-transcriptional control of gene expression. Through systematic perturbations of positioning and stoichiometry, we also demonstrate the sensitivity of our assay in detecting regulatory activity of protein domains in low copy number and in both 5' and 3' UTRs. We identify some domains that can downregulate gene expression both when recruited to DNA and RNA, and dissect their mechanisms of regulation. Finally, we build a new synthetic RNA regulator, synNANOS, that can stably maintain gene expression at desired levels that are predictable by a mathematical model for post-transcriptional control of gene expression. This work serves as a resource for human RNA regulatory domains that are separate from RNA-binding domains, and expands the synthetic repertoire of RNA-based genetic control tools and high-throughput assays.