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 in silico selection, in vitro 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.