Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways

TLDR

In mammals, miRNAs and siRNAs are loaded into the same RISC to guide mRNA degradation or translation silencing, while in Drosophila AGO2 is part of RISC and AGO1 associates with Dicer‑1 and pre‑miRNA, suggesting a role in miRNA biogenesis. The study demonstrates that AGO2 is essential for siRNA‑directed RNAi, required for siRNA duplex unwinding and RISC assembly in Drosophila embryos. Loss of AGO2 abolishes siRNA‑directed RNAi but leaves miRNA‑directed cleavage intact, whereas AGO1 is dispensable for siRNA cleavage yet necessary for mature miRNA production and miRNA‑directed cleavage, indicating distinct Argonaute proteins act at different steps of small RNA silencing and revealing inherent differences between siRNA‑ and miRNA‑initiated RISCs in Drosophila.

Abstract

In mammalian cells, both microRNAs (miRNAs) and small interfering RNAs (siRNAs) are thought to be loaded into the same RNA-induced silencing complex (RISC), where they guide mRNA degradation or translation silencing depending on the complementarity of the target. In Drosophila, Argonaute2 (AGO2) was identified as part of the RISC complex. Here we show that AGO2 is an essential component for siRNA-directed RNA interference (RNAi) response and is required for the unwinding of siRNA duplex and in consequence assembly of siRNA into RISC in Drosophila embryos. However, Drosophila embryos lacking AGO2, which are siRNA-directed RNAi-defective, are still capable of miRNA-directed target RNA cleavage. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, which is dispensable for siRNA-directed target RNA cleavage, is required for mature miRNA production that impacts on miRNA-directed RNA cleavage. The association of AGO1 with Dicer-1 and pre-miRNA also suggests that AGO1 is involved in miRNA biogenesis. Our findings show that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism and suggest that there are inherent differences between siRNA-initiated RISCs and miRNA-initiated RISCs in Drosophila.