Large-scale tethered screen of RNA-binding proteins reveals novel regulators of poly(A) site selection [RNA-Seq]

Alternative polyadenylation (APA) is a widespread mechanism of gene regulation that generates transcript isoforms with distinct 3' ends. While several RNA-binding proteins (RBPs) are known to regulate APA, the roles of the vast majority of RBPs in this process remain poorly characterized. Using a large-scale tethered function assay, we screened a library of 879 human RBPs to identify regulators of APA. We discovered 52 RBPs that modulate APA, validated their functions through knockdown PAS-seq and enhanced crosslinking and immunoprecipitation (eCLIP), and developed a fine-tuned transformer model to predict activators of PAS selection. We focused on two unexpected hits: GRB2, an adaptor protein with crucial roles in cell signaling, and RNPS1, a member of the exon junction complex (EJC). We uncovered mechanisms by which they modulate APA using affinity purification mass spectrometry (AP-MS) and biochemical assays. Our screen provides a comprehensive resource of RBPs involved in APA and reveals unexpected roles of signaling factors in APA regulation.