Small-RNA asymmetry is directly driven by mammalian Argonautes. Mus musculus

Asymmetric selection of single-stranded guide RNAs from double-stranded RNA (dsRNA) precursors is crucial for RNA silencing-mediated gene regulation. However, the precise mechanisms for small RNA asymmetry remain unclear, especially since asymmetric selection can still occur under depletion of putative asymmetry sensors, Drosophila R2D2 and mammalian Dicer. Here we report direct contribution of mammalian Argonaute 2 (Ago2) to microRNA (miRNA) asymmetry. Ago2 selects strands with 5´-uridine/adenosine and thermodynamically unstable 5´-ends in parallel through its two sensor regions, which contact 5´-nucleobase and 5´-phosphate(s) of the prospective guide strands, respectively. Consistently, miRNA asymmetry shows characteristic digital-analog superposed patterns reflecting 5'-end nucleotide identity and thermodynamic stability. Furthermore, we demonstrate that cancer-associated miRNA variations reprogram asymmetric selection. Finally, our study presents a model of this universal principle that will aid a comprehensive understanding of miRNA function and therapeutic reinvention of RNA silencing in precision medicine. Overall design: Immunoprecipitation of WT or mutant Ago2 in mouse ES cells (Ago-null background) and small RNA sequencing