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

RNA regulation plays an integral role in tuning gene expression and is controlled by thousands of RNA-binding proteins (RBPs). While some RBPs require most of their sequence to accomplish their functions, some examples exist of modularity: small protein domains that serve as molecular glues to recruit larger RNA regulatory complexes. Mapping these RNA-regulatory effector domains is important for understanding RBP function and for developing compact synthetic tools for precisely tuning RNA levels. We developed a high-throughput recruitment assay (HT-RNA-Recruit) to identify RNA-down-regulatory effector domains within human RBPs. By recruiting over 30,000 protein tiles from 367 RBPs to a reporter mRNA, we discovered over 100 unique RNA-down-regulatory effector domains in 86 distinct RBPs. We identified some domains that downregulate gene expression both when recruited to DNA and RNA, and dissected their mechanisms of regulation. Finally, we built inducible synthetic RNA regulators that can target endogenous RNAs or stably maintain reporter gene expression at desired levels, and developed a mathematical model to predict their dynamics. This work serves as a resource for human RNA regulators and expands the synthetic repertoire of RNA-based genetic control tools. 1. High-throughput recruitment assay to measure the effects on reporter RNA expression from tethering a 30,000 member or a smaller, 3,000 member library to different reporter constructs: 24 MS2 stem-loops in the 3' UTR or 5'UTR; 7 MS2 stem-loops in the 5' UTR; 9xTetO binding sites at a proximal enhancer 2. RNA-seq profiling of K562 cells after 48 hours of overexpression of either full-length RBPs, MCP alone, full-length RBP mutants, or a synthetic RNA repressor with an endogenous RBD 3. CUT&RUN profiling of the reporter locus after DNA recruitment of ZNF10 KRAB, RNA recruitment of an RNA regulator, or RNA recruitment of ZNF10 KRAB