In Drosophila, miRNA strands are predominantly sorted into AGO1 to regulate seed-matched target transcripts, while their partner miRNA∗ strands are thought to be mostly degraded. Here, we report that Drosophila Argonautes exhibit different strand preferences for miRNA duplexes, and that in particular, many miRNA∗ species accumulate in the RNAi effector AGO2. AGO2-loaded miRNA∗ species require canonical RNAi factors for their accumulation, are efficiently 3′ modified, and are preferentially active on extensively matched target transcripts. Differential miRNA/miRNA∗ sorting profiles are correlated with specific central mismatches. In vitro assays revealed an active role for Watson-Crick base-pairing at positions 9 and 10 in promoting strand selection by AGO2, with little reciprocal effect on strand selection by AGO1. We conclude that miRNA strand selection and sorting are actually linked processes that stem from distinct loading preferences of AGO proteins and that independent sorting of duplex strands is a general feature of Drosophila microRNA genes.
