A protein sensor for siRNA asymmetry. Science

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Science (Impact Factor: 33.61). 12/2004; 306(5700):1377-80. DOI: 10.1126/science.1102755
Source: PubMed


To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.

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    • "siR- NAs are 21-nt long duplex RNAs that are bound by a heterodimer between Dcr-2 and r2d2, a dsRBP partner [28]. The Dcr-2/r2d2 complex transfers the duplex siRNA to Argonaute-2 (AGO2) to form siRNA programmed RISC (siRISC) [29] [30]. Mature siRISC that carries only the guide strand of the siRNA duplex is formed after AGO2 cleaves the passenger strand that is then released and further degraded [31] [32]. "
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    • "With the help of a nuclear transport receptor (exportin-5) and Ran-GTP, pre-miRNAs are transported from nucleus to cytoplasm [22, 23]. In the cytoplasm, pre-miRNAs are recognized by Dicer, which works in cooperation with human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA binding protein (TRBP or Loquacious in Drosophila) to cleave pre-miRNAs into miRNA duplexes [24–28]. Together with Argonaute and other proteins, a miRNA duplex is then loaded to generate RISC [29–31]. "
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    • "Recent studies of the molecular mechanism that underly the specificity of the RNAi machinery indicate that the protein complex that mediate the recognition of the target RNA by the short dsRNA are not symmetrical. Thus, via mechanisms that are not fully understood, RISC treats only one of the strands as a guide (Tomari et al., 2004; Rand et al., 2005; Betancur and Tomari, 2012; Noland and Doudna, 2013). It is likely that a similar mechanism is at play here. "
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