Argonaute Loading Improves the 5′ Precision of Both MicroRNAs and Their miRNA∗ Strands in Flies

SummaryMicroRNAs (miRNAs) are short regulatory RNAs that direct repression of their mRNA targets. The miRNA “seed”—nucleotides 2–7—establishes target specificity by mediating target binding 1, 2, 3, 4 and 5. Accurate processing of the miRNA 5′ end is thought to be under strong selective pressure 6 and 7 because a shift by just one nucleotide in the 5′ end of a miRNA alters its seed sequence, redefining its repertoire of targets (Figure 1). Animal miRNAs are produced by the sequential cleavage of partially double-stranded precursors by the RNase III endonucleases Drosha and Dicer, thereby generating a transitory double-stranded intermediate comprising the miRNA paired to its partially complementary miRNA∗ strand 8 and 9. Here, we report that in flies, the 5′ ends of miRNAs and miRNA∗ strands are typically more precisely defined than their 3′ ends. Surprisingly, the precision of the 5′ ends of both miRNA and miRNA∗ sequences increases after Argonaute2 (Ago2) loading. Our data imply that either many miRNA∗ sequences are under evolutionary pressure to maintain their seed sequences—that is, they have targets—or that secondary constraints, such as the sequence requirements for loading small RNAs into functional Argonaute complexes, narrow the range of miRNA and miRNA∗ 5′ ends that accumulate in flies.