A resource of RNA-binding protein motifs across eukaryotes reveals evolutionary dynamics and gene-regulatory function

Although RNA-binding proteins (RBPs) are key regulators of gene expression, their RNA-binding specificities, i.e., motifs, have not been comprehensively determined. Here, we introduce EuPRI (Eukaryotic Protein-RNA Interactions), a freely available resource of RNA motifs for 34,746 RBPs from 690 eukaryotes. EuPRI includes in vitro binding data for 504 RBPs, including newly collected RNAcompete data for 174 RBPs, along with thousands of predicted motifs. We predict these motifs with a new algorithm, Joint Protein-Ligand Embedding (JPLE), which can detect distant homology relationships and map specificity-determining peptides. EuPRI quadruples the number of available RBP motifs, expanding the motif repertoire across all major eukaryotic clades, and assigning motifs to the majority of human RBPs. We demonstrate the utility of EuPRI for inferring post-transcriptional function and evolutionary relationships by identifying rapid, recent evolution of post-transcriptional regulatory networks in worms and plants, in contrast to the vertebrate RNA motif set, which has remained relatively stable after a large expansion between metazoan and vertebrate ancestors. Here, we analyze the RNA-binding preferences of 174 diverse eukaryotic RNA-binding proteins using RNAcompete. In the RNAcompete assay, purified GST-tagged RNA-binding protein is incubated with an excess of RNA pool and bound RNA from individual pulldown experiments are directly labeled and hybridized to a custom Agilent 244K microarray.