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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Available from: Stefan Ameres, Oct 10, 2015
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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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    ABSTRACT: Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools—referred to as siPools—containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects.
    Nucleic Acids Research 05/2014; 42(12). DOI:10.1093/nar/gku480 · 9.11 Impact Factor
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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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    ABSTRACT: microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small RNAs that repress gene expression at the post-transcriptional level in plants and animals. Small RNAs guide Argonaute-containing RNA-induced silencing complexes to target RNAs in a sequence-specific manner, resulting in mRNA deadenylation followed by exonucleolytic decay, mRNA endonucleolytic cleavage, or translational inhibition. Although our knowledge of small RNA biogenesis, turnover, and mechanisms of action has dramatically expanded in the past decade, the subcellular location of small RNA-mediated RNA silencing still needs to be defined. In contrast to the prevalent presumption that RNA silencing occurs in the cytosol, emerging evidence reveals connections between the endomembrane system and small RNA activities in plants and animals. Here, we summarize the work that uncovered this link between small RNAs and endomembrane compartments and present an overview of the involvement of the endomembrane system in various aspects of RNA silencing. We propose that the endomembrane system is an integral component of RNA silencing that has been long overlooked and predict that a marriage between cell biology and RNA biology holds the key to a full understanding of post-transcriptional gene regulation by small RNAs.
    The EMBO Journal 05/2014; 33(9). DOI:10.1002/embj.201387262 · 10.43 Impact Factor
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    • "Synthetic siRNAs can also be introduced into cells in order to experimentally activate RNAi [2]. siRNA duplexes with 5 í® í° -hydroxyl ends are rapidly phosphorylated in cells by the cellular kinase Clp1 [5], then the siRNA strand with the thermodynamically less stable 5 í® í° end is preferentially incorporated as the guiding or antisense strand (AS) in the RNA-induced silencing complex (RISC) [6], while the passenger or sense strand (SS) of the siRNA duplex is cleaved by the human Argonaute 2 protein (hAgo 2) and liberated from the complex [7]. The selection of the guide strand is then based on the thermodynamic stability of the siRNA duplex ends, the strand that is always the one whose 5 í® í° end is less tightly paired to its complement. "
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    ABSTRACT: The active components of the RNAi are 21 nucleotides long dsRNAs containing a 2 nucleotide overhang at the 3' end, carrying 5'-phosphate and 3'-hydroxyl groups (siRNAs). Structural analysis revealed that the siRNA is functionally bound at both ends to RISC. Terminal modifications are considered with interest as the introduction of chemical moieties interferes with the 3' overhang recognition by the PAZ domain and the 5'-phosphate recognition by the MID and PIWI domains of RISC. Herein, we report the synthesis of modified siRNAs containing terminal amide linkages by introducing hydroxyethylglycine PNA (hegPNA) moieties at 5', and at 3' positions and on both terminals. Results of gene silencing studies highlight that some of these modifications are compatible with the RNAi machinery and markedly increase the resistance to serum-derived nucleases even after 24 h of incubation. Molecular docking simulations were attained to give at atomistic level a clearer picture of the effect of the most performing modifications on the interactions with the human Argonaute 2 PAZ, MID, and PIWI domains. This study adds another piece to the puzzle of the heterogeneous chemical modifications that can be attained to enhance the silencing efficiency of siRNAs.
    03/2014; 2014(1):901617. DOI:10.1155/2014/901617
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