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

Alternative polyadenylation (APA) generates transcript isoforms with distinct 3' ends, yet the repertoire of its protein regulators remains poorly defined. Using a large-scale tethered function screen, we profiled 879 human RNA-binding proteins (RBPs) and identified 63 high-confidence activators of poly(A) site (PAS) selection, most of which were not previously linked to APA. We validated these factors by knockdown PAS-seq, RNA sequencing (RNA-seq), and enhanced cross-linking and immunoprecipitation (eCLIP) analyses and developed a fine-tuned protein language model that predicts PAS selection activators and their key functional domains. We then mechanistically dissected two unexpected hits: GRB2, a signaling adaptor protein, and RNPS1, a peripheral component of the exon junction complex (EJC). Both regulate APA, at least in part, through direct interactions with distinct subunits of the cleavage and polyadenylation (CPA) machinery. Together, our study provides a comprehensive resource of APA-regulating RBPs and uncovers unexpected roles of signaling and EJC factors in APA regulation.