G-quadruplexes in the proximity 3′-UTR enhances alternative polyadenylation of Neogenin 1

Polyadenylation is a crucial step in mRNA maturation. Fifty percent of the time alternative polyadenylation sites (PASs) are used instead of canonical ones, affecting the length of the transcripts' 3'-UTRs. The mechanisms controlling the selection of these alternative PASs remain unknown. Using a unique sequencing method (PolyA Click-seq) to identify different polyadenylated isoforms, PolyA events modulated by RHPS4, a ligand known to stabilize RNA G-quadruplex structures (rG4s), were revealed. Through <i>in silico</i> selection, <i>in vitro</i> assays, and rG4 mutagenesis, the pivotal role of rG4 structures in determining PASs was uncovered, most notably in the case of the gene encoding Neogenin-1 (NEO1), a cell surface protein that is a member of the immunoglobulin superfamily. Our findings highlight the importance of rG4-mediated alternative polyadenylation (APA) regulation in the 3'-UTR as a method to both alter isoform choice and impact protein synthesis, with potential relevance for RNA-based therapeutics.