Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep 7:314-320

Institute of Molecular Biotechnology of the Austrian Academy of Sciences, IMBA, Dr-Bohr-Gasse 3-5, 1030 Vienna, Austria.
EMBO Reports (Impact Factor: 9.06). 04/2006; 7(3):314-20. DOI: 10.1038/sj.embor.7400637
Source: PubMed


A crucial step in the RNA interference (RNAi) pathway involves the assembly of RISC, the RNA-induced silencing complex. RISC initially recognizes a double-stranded short interfering RNA (siRNA), but only one strand is finally retained in the functional ribonucleoprotein complex. The non-incorporated strand, or 'passenger' strand, is removed during the assembly process and most probably degraded thereafter. In this report, we show that the passenger strand is cleaved during the course of RISC assembly following the same rules established for the siRNA-guided cleavage of a target RNA. Chemical modifications impairing the cleavage of the passenger strand also impair the cleavage of a target RNA in vitro as well as the silencing of a reporter gene in vivo, suggesting that passenger strand removal is facilitated by its cleavage during RISC assembly. Interestingly, target RNA cleavage can be rescued if an otherwise non-cleavable passenger strand shows a nick at the scissile phosphodiester bond, which further indicates that the cleavage event per se is not essential.

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    • "miRNAs and siRNAs have distinct requirements for mRNA target recognition. The base pairing between miRNAs and their targets is degenerate, whereas the pairing between siRNAs and their targets requires high fidelity, typically allowing no more than three mismatches (Leuschner et al., 2006; Watanabe et al., 2008). Localization of the miRNAs is theoretically unpredictable today. "
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    • "Although these proteins are very similar, only Ago2 is endonucleolytically active (12,13). Ago2 not only cleaves the complementary target RNA but uses its cleavage activity also during RISC loading: Ago2 binds to the ds siRNA and cleaves the passenger strand leading to faster and more efficient loading of the siRNA into Ago2-containing RISC complexes (14–16). Although less efficient, non-catalytic Ago proteins load siRNA guide strands in RNAi experiments (17,18) and it has been reported that non-catalytic Ago proteins can contribute to the knockdown as well (19). "
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    • "RISC is recruited to targets through base pairing between the miRNA or siRNA guide and the target transcript, resulting in target gene repression. For siRISC in both plants and animals, the siRNA is fully complementary to the target transcript and siRISC cleaves the target transcript through the endonuclease activity of the AGO protein, such as atAGO1 in Arabidopsis and Ago2 in mammals (Baumberger & Baulcombe, 2005; Matranga et al, 2005; Rand et al, 2005; Leuschner et al, 2006). For miRISC, the mode of target recognition is different between plants and animals. "
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