Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins

Abstract

Until now, determining the sequences recognized by an RNA-binding protein has been time and labor intensive. Ray et al. use a custom pool of >210,000 oligos that encode linear and stem-loop RNAs to rapidly determine the sequences bound by nine RNA-binding proteins. Metazoan genomes encode hundreds of RNA-binding proteins (RBPs) but RNA-binding preferences for relatively few RBPs have been well defined1. Current techniques for determining RNA targets, including in vitro selection and RNA co-immunoprecipitation2,3,4,5, require significant time and labor investment. Here we introduce RNAcompete, a method for the systematic analysis of RNA binding specificities that uses a single binding reaction to determine the relative preferences of RBPs for short RNAs that contain a complete range of k-mers in structured and unstructured RNA contexts. We tested RNAcompete by analyzing nine diverse RBPs (HuR, Vts1, FUSIP1, PTB, U1A, SF2/ASF, SLM2, RBM4 and YB1). RNAcompete identified expected and previously unknown RNA binding preferences. Using in vitro and in vivo binding data, we demonstrate that preferences for individual 7-mers identified by RNAcompete are a more accurate representation of binding activity than are conventional motif models. We anticipate that RNAcompete will be a valuable tool for the study of RNA-protein interactions.