A role for human Dicer in pre-RISC loading of siRNAs

Abstract

RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3′ overhangs and the thermodynamic properties of 2–4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

“…51 In contrast, the protein has been shown to directly bind small interfering (si)RNAs. [52][53][54] By electrophoresis mobility shift assay (EMSA), using a radiolabelled control siRNA and recombinant MBP or MBP-TRBP, we observed several low mobility bands, which indicates that TRBP binds to the siRNA either as a monomer or as a multimer ( Fig. 2A, left).…”

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…The sncRNA-duplex/AGO-2 complex is called pre-RISC (pre-RNA-induced silencing complex) and requires the removal of one of the strands of the RNA, the passenger strand, in order to become an active RISC complex [81,82] (Figure 1). The strand that remains in RISC is known as the guide strand.…”

Dicer is dispensable for asymmetric RISC loading in mammals

“…It was previously reported that, in mammalian cell lysate, siRNA duplexes form a complex containing Dicer and TRBP (therein referred to as complex D or pre-RLC), which can be separated on polyacrylamide native gel (Pellino et al 2005;Sakurai et al 2011). Indeed, we readily detected such a complex D with radiolabeled let-7 siRNA duplex in WT MEF lysate and Dicer1 À/À MEF lysate supplemented with rDICER1 ( Fig.…”

“…Native gel analysis, UV crosslinking, dicing, and target cleavage assays Methods were previously described in detail and were used with minor modifications (Haley et al 2003;Pellino et al 2005;Yoda et al 2010;Kawamata and Tomari 2011;Sakurai et al 2011). In sum, in vitro RISC assembly reactions typically contained 3 mL 403 reaction mix, 10 nM target RNA, 10 nM radiolabeled RNA duplex, 4 mL cell lysate, and 1 mL rDICER1 in 10 mL reaction mixture.…”

“…By analogy to the functions of fly Dcr-2/R2D2 in thermodynamic asymmetry sensing and Ago2-RISC loading, it is tempting to postulate that Dcr-1 and Loqs in flies and Dicer and TRBP (or PACT) in mammals might play similar roles in RISC assembly, in addition to their essential role in dicing Gregory et al 2005;Maniataki and Mourelatos 2005;MacRae et al 2008;Miyoshi et al 2009;Sakurai et al 2011). Indeed, it was recently shown that, much as fly Dcr-2/R2D2, recombinant human Dicer/ TRBP can bind to siRNA duplexes according to their thermodynamic asymmetry (Gredell et al 2010;Noland et al 2011).…”