3’ Nucleotide Asymmetry Directs miRNA Strand Selection

Accurate microRNA (miRNA) strand selection is essential for defining the regulatory landscape of the miRNA-induced silencing complex (miRISC). While 5′ nucleotide identity and duplex thermodynamics have been proposed to bias strand choice, these features cannot fully explain in vivo strand preferences. Here, we uncover a conserved and previously unrecognized role for 3′ nucleotide asymmetry in directing miRNA strand selection in Caenorhabditis elegans and human cells. A favorable 3′ terminal nucleotide on the passenger strand promotes selective loading of the opposing guide strand into miRISC, revealing a cooperative interplay between 5′ and 3′ terminal asymmetries that ensures precise strand discrimination. These findings establish a unified, evolutionarily conserved mechanism for miRNA duplex sorting and expand the fundamental rules governing small RNA biogenesis.