Endogenous small interfering RNAs (endo-siRNAs) in Drosophila are processed by Dicer-2 (Dcr-2) and loaded onto Ago2 by the Dcr-2/R2D2 heterodimer. In r2d2 mutants, the level of endo-siRNAs is unchanged, but endo-siRNAs are misloaded onto Ago1. However, the mechanism underlying the control of endo-siRNA sorting by R2D2 remains unknown. Here, we show that R2D2 controls endo-siRNA sorting by localizing Dcr-2, and presumably endo-siRNA duplexes, to cytoplasmic foci, D2 bodies. Ago2, but not Ago1, localized to D2 bodies. dsRNA-binding-deficient mutant, but not wild-type, R2D2 failed to localize D2 bodies and caused endo-siRNA misdirection to Ago1 in R2D2-depleted cells. However, R2D2 was dispensable for sorting miRNAs and exogenous siRNAs onto Ago1 and Ago2, respectively, in vivo. Endo- and exo-siRNA guide selection also occurred R2D2 independently. The functions of R2D2 are required to avoid endo-siRNA misdirection to Ago1, because Ago1 is capable of loading incompletely complementary miRNA duplexes and endo-siRNA duplexes.
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