siRNA Repositioning for Guide Strand Selection by Human Dicer Complexes

Abstract

SUMMARY The human ribonuclease Dicer and its double-stranded RNA (dsRNA) binding protein (dsRBP) partners TRBP and PACT play important roles in the biogenesis of regulatory RNAs. Following dicing, one dsRNA product strand is preferentially assembled into an RNA-Induced Silencing Complex (RISC). The mechanism of strand selection in humans and the possible role of Dicer in this process remains unclear. Here we demonstrate that dsRNAs undergo significant repositioning within Dicer complexes following dicing. This repositioning enables directional binding of RNA duplexes, thereby biasing their orientation for guide strand selection according to the thermodynamic properties of the helix. Our findings indicate that Dicer is itself capable of sensing siRNA thermodynamic asymmetry regardless of the dsRBP to which it is bound. These results support a model in which Dicer employs two distinct RNA binding sites – one for dsRNA processing and the other for sensing of siRNA thermodynamic asymmetry – during RISC loading in humans.

ATP-independent diffusion of double-stranded RNA binding proteins

“…In other words, Dicer-TRBP diffusion can serve to scan presiRNA and pre-miRNA substrates and to facilitate Dicer's catalytic activity by locating the complex at the RNA cleavage site with an improved efficiency and precision (46,47). It is also likely that TRBP's diffusion plays another role in RNA-mediated gene silencing, for instance a recruitment of Dicer-TRBP complex to Ago2 (10) or guide strand selection by dsRNA scanning (48,49).…”

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

“…This ability appears to be activated through its interaction with Transactivation Response (TAR) RNABinding Protein (TRBP) and Protein Kinase RNA (PKR) Activator (PACT), both double-stranded RNA binding proteins (dsRBP) [85] ( Figure 1). However, there is contradictory evidence regarding this role for Dicer [85,86] , and further research may be needed to clarify these observations. Nonetheless, it is important to mention that proper selection of the guide ensures specificity towards silencing the intended target.…”

MicroRNAs in the Atherosclerotic Plaque

“…Analysis has shown that the strand with less stable base pair binding in its 5Ј end is more often selected as a guide strand. It has been suggested that the Dicer is repositioned after the cleavage reaction and, in its new position, the helicase domain in Dicer senses the thermodynamic stability of the ends and incorporates the guide strand into the RISC (forming miRISC) (12 ).…”