21, 5875-85 Dicer siRNA generation kit (Gene Therapy Systems, Inc., San Diego, Calif., Catalog No.T51001). Recombinant Dicer proteins have been used to make siRNA see, Myers et al., 2003, Nature Biotechnol 21, 324-8 Beach et al., 2003, US Pat Appl Publ US2003/0084471 Zhang et al., 2002, EMBO J. Our data reveal that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating the passage of siRNA from Dicer to RISC. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and facilitates its incorporation into RISC. Association with R2D2 does not affect the siRNA-generating activity of DCR-2. Tandem dsRNA-binding domains is a feature shared by the C. The purified enzyme comprises two stoichiometric subunits, DCR-2 and a novel protein that we named R2D2 because it contains two dsRNA-binding domains (R2) and is associated with DCR-2 (D2). Here we disclose the biochemical purification of siRNA-generating activity. The two steps of the RNAi pathway appear to be closely coupled because the direction of dsRNA processing determines which strand of siRNA becomes the guiding strand for RISC (11). It remains unclear how the siRNA generated by Dicer becomes incorporated into RISC. Two Dicer enzymes, DCR-1 and DCR-2, have been identified in Drosophila (5). The endonuclease that processes dsRNA has been identified as Dicer, a family of large non-canonical RNase III enzymes (5). Secondly, the siRNA is incorporated into a nuclease complex known as RNA-initiated silencing complex (RISC), and functions as a guide RNA to direct RISC-mediated sequence-specific mRNA degradation (6, 9-11). First, long dsRNA molecules are cleaved into 21 to 23-nucleotide (nt) small interfering RNA (siRNA) duplexes (5-8). Emerging evidence indicates that the RNAi and related pathways function in many fundamental biological processes including antiviral defense, development, and maintenance of genomic stability (4).Įxperiments in the model Drosophila system indicate that the RNAi pathway consists of the initiation and effector steps. The biological importance of RNAi is underscored by its wide conservation throughout metazoans and the existence of closely related systems in plants (co-suppression) and fungi (quelling) (4). RNA interference (RNAi) is a form of post-transcriptional gene silencing whereby dsRNA molecules trigger the sequence-specific degradation of cognate messenger RNA (mRNA) (1-3). The field of this invention is an enzyme of RNA silencing.
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