A 5′-uridine amplifies miRNA/miRNA* asymmetry in Drosophila by promoting RNA-induced silencing complex formation

Our analyses of fly, worm and mouse miRNA sequence data reveal that the 5′-U is recognized after miRNA production. Only one of the two strands can be assembled into Argonaute protein from a single miRNA/miRNA* molecule: in fly embryo lysate, a 5′-U promotes miRNA loading while decreasing the loading of the miRNA*.We suggest that recognition of the 5′-U enhances Argonaute loading by a mechanism distinct from its contribution to weakening base pairing at the 5′-end of the prospective miRNA and, as recently proposed in Arabidopsis and in humans, that it improves miRNA precision by excluding incorrectly processed molecules bearing other 5′-nt.MicroRNA (miRNA) are approximately 22-nt regulatory RNA that direct members of the Argonaute protein family to their mRNA targets [1]. Together, miRNA guide and the Argonaute protein form the core of the RNA-induced silencing complex (RISC), which recognizes its mRNA targets primarily through its seed sequence, nt 2 through nt 7 [2].The RNase III enzymes Drosha and Dicer excise most animal miRNA from long primary transcripts (pri-miRNA). Drosha cleaves pri-miRNA to release an approximately 65-nt pre-miRNA; Dicer cleaves the pre-miRNA to liberate a miRNA/miRNA* duplex. The duplex is then loaded into an Argonaute protein. The geometry of the miRNA/miRNA* duplex during the loading reaction determines the fate of each small RNA: the miRNA binds tightly to Argonaute, with its 5′-nt anchored in a positively charged pocket in the Mid domain of the protein [3,4]. The miRNA* assumes the same position as subsequent mRNA targets and is held to the complex predominantly by seed sequence base pairing. A seed sequence mismatch between the miRNA and its miRNA* is believed to promote miRNA* dissociation [5,6]. A subset of Argonaute proteins can cleave the miRNA* if it is extensively paired to the miRNA, triggering its destruction [7-10]. The orientation of the duplex during Argonaute loading is not random: the miRNA is usually the strand with the