Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are conserved families of small regulatory RNAs (11
) that operate within related molecular pathways (16
). In both cases, cytoplasmic Dicer class RNase III enzymes metabolize double-stranded RNA (dsRNA) precursors into small RNA duplexes. These mature into single-stranded RNA associated with an Argonaute (AGO) protein in an RNA-induced silencing complex (RISC), which is guided by the small RNA to target transcripts. Animal miRNAs have an additional preceding processing step in which primary miRNA transcripts are cleaved by the nuclear RNase III enzyme Drosha, yielding ~60- to 70-nucleotide (nt) pre-miRNA hairpins that serve as Dicer substrates (29
RNase III enzymes often require dsRNA binding protein (dsRBP) cofactors. Drosophila
Drosha binds directly to the dsRBP Pasha, which is orthologous to mammalian DGCR8; this family is essential for pre-miRNA generation in all animals (57
encodes two Dicers, of which the miRNA processing enzyme Dcr-1 binds the dsRBP Loquacious (Loqs)-PB isoform to generate miRNA duplexes (29
). Curiously, a distinct isoform, Loqs-PD, binds the siRNA-generating enzyme Dcr-2 and is required for endogenously encoded siRNA (endo-siRNA) biogenesis (19
). Another dsRBP, R2D2, also binds Dcr-2 and is required to load siRNAs into AGO2. The R2D2/Dcr-2 heterodimer is the key constituent of the RISC loading complex (RLC), which determines dominant guide strands by sensing thermodynamic asymmetry of highly paired small RNA duplexes (35
). In particular, the duplex strand whose 5′ end resides in the more unstable end is usually selected as the mature guide strand (28
). The single mammalian Dicer binds the dsRBPs TRBP and PACT, which are also involved in RISC loading (29
Despite their parallels, the miRNA and siRNA pathways are substantially separated in Drosophila
). Their small RNAs are not only generated by different Dicers but also sorted into functionally distinct Argonautes. miRNAs accumulate preferentially in AGO1, which is specialized for its capacity to repress targets with limited complementarity, as little as 6 to 7 nucleotides of base pairing to miRNA 5′ ends (positions 2 to 7 or 2 to 8). siRNAs accumulate preferentially in AGO2, which exhibits greater enzymatic cleavage activity toward highly or perfectly complementary targets. Since the siRNA/AGO2 pathway is specifically exploited when the RNA interference (RNAi) technique is used, it is relevant to elucidate AGO2 cargoes and understand mechanisms that ensure correct siRNA loading and function.
The first known endogenous function of Drosophila
RNAi was to defend against RNA viruses (1
). In addition, endo-siRNAs are generated from transposable elements (TEs), overlapping transcripts (cis
-natural antisense transcripts [cis
-NATs]), and structured loci (hairpin RNAs [hpRNAs]) to regulate expression of TEs and mRNA targets (8
). Recently, miRNA* strands, the partner strands of mature miRNAs, were recognized as a substantial source of AGO2-loaded small RNAs (10
). Building upon earlier mechanistic work (13
), these studies documented that AGO1 and AGO2 have distinct strand preferences according to 5′ nucleotide identity and central structure in the precursor duplex. In particular, AGO2 strongly prefers the strand whose 9th and 10th nucleotides from the 5′ end are paired, and this structural preference can override thermodynamic asymmetry.
Because mutants of siRNA pathway components seem to exhibit little or no RNAi activity (32
), it is usually assumed that the miRNA pathway cannot incorporate siRNAs as guide molecules. Although there is competition between AGO1 and AGO2 loading machineries (13
), a UV cross-linking assay showed that AGO1 can largely reject synthetic siRNAs even in the absence of AGO2 loading machinery (55
). On the other hand, genetics suggested possible interactions between these pathways, since double mutants of miRNA and siRNA pathway genes exhibited synthetic phenotypes (24
). Moreover, the sorting of miR-277, whose mature strand resides in both AGO1 and AGO2 complexes, is altered by the availability of the two Argonautes (13
). Taken together, it is not clear how broadly such flexible sorting applies. In addition, a mechanism to sense central duplex mismatch positions, postulated to underlie the distinct strand preference of AGO2 and AGO1 for miRNA/miRNA* duplex strands, has not been elucidated.
In this study, we focused our attention on the roles of R2D2 in small RNA sorting. Analysis of AGO2-associated small RNAs showed an essential role of R2D2 in endogenous small RNA loading to AGO2. Furthermore, all classes of endo-siRNAs, but specifically not miRNA* species, were bound more efficiently by AGO1 in r2d2 and ago2 mutants than in the wild type. Functional consequences for endo-siRNA misdirection into AGO1 in r2d2 mutants were demonstrated, and in vitro assays demonstrated that the R2D2/Dcr-2 complex is directly involved in sensing central mismatch positions to determine the strand for AGO2 loading. Together, these results establish new roles of R2D2 in small RNA-mediated gene regulatory networks.