Small RNA Structure and Dicer/Argonaute Dependence During RISC Assembly in DrosophilaPublic Deposited
Short interfering RNAs (siRNAs) and microRNAs (miRNAs) are both processed from longer, double-stranded RNA (dsRNA) presursors by a member of the Dicer (Dcr) family of proteins. siRNAs generally arise from perfectly base-paired dsRNAs, whereas miRNAs are excised from 60-70 nt pre-miRNA hairpins containing multiple bulges and mismatches within the duplexed region. In animals, siRNAs usually induce target mRNA endonucleolytic cleavage, whereas miRNAs usually direct translation inhibition or exonucleolytic degradation. The Argonaute (Ago) proteins associate directly with mature small RNAs within RNA-induced silencing complexes (RISCs). When RISC directs silencing via endonucleolytic mRNA cleavage, the small-RNA-directed "slicer" activity resides within the Argonaute protein itself. The Drosophila siRNA and miRNA pathways involve functionally specialized isoforms of Dicer and Argonaute proteins. Previous studies showed that Dcr-1 and Ago-1 are primarily involved in miRNA biogenesis and silencing, whereas Dcr-2 and Ago-2 are mostly devoted to the siRNA pathway. A more recent model suggested Ago-2 is involved in the functioning of some miRNAs, and Dcr-2 acts as a gatekeeper for the assembly of Ago-2 mediated RNA-induced silencing complexes (RISCs) by promoting the incorporation of siRNAs and disfavoring miRNAs as loading substrates for Ago-2. Our study used the differential Dicer-2 dependence exhibited by siRNA and miRNA duplexes in vitro to define RNA features that dictate Dcr-2 specificity. We found that the degree of base pairing at the 5' end of the guide strand plays a predominant role in specifying siRISC assembly. We also found unanticipated variety in the Dcr-2 and Ago-2 dependence displayed by a series of miRNAs with different structural features, some of which disagree with current models. These data, combined with other studies, suggest that miRNAs may possess less uniformity than previously envisioned in their functional characteristics.