Abstract

During microRNA (miRNA) biogenesis, one strand of a |[sim]|21–22-nucleotide RNA duplex is preferentially selected for entry into a silencing complex. The other strand, known as the miRNA|[ast]| species, has typically been assumed to be a carrier strand. Here we show that, although Drosophila melanogaster miRNA|[ast]| species are less abundant than their partners, they are often present at physiologically relevant levels and can associate with Argonaute proteins. Comparative genomic analyses revealed that >40% of miRNA|[ast]| sequences resist nucleotide divergence across Drosophilid evolution, and at least half of these well-conserved miRNA|[ast]| species select for conserved 3|[prime]| untranslated region seed matches well above background noise. Finally, we validated the inhibitory activity of miRNA|[ast]| species in both cultured cells and transgenic animals. These data broaden the reach of the miRNA regulatory network and suggest an important mechanism that diversifies miRNA function during evolution.