The Role of Human Dicer-dsRBD in Processing Small Regulatory RNAs

Abstract

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded – double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

“…GST alone did not bind any of the tested dsRNAs (data not shown). Recently, another study reported the RNA-binding characteristics of the human Dicer dsRBD as an isolated domain, comparing it to the first dsRBD of DGCR8 (Wostenberg et al 2012). The reported K d values for Dicer-dsRBD ranged from ∼2 to 10 μM, with the affinity of binding decreasing with decrease of the substrate length, which is similar to our findings.…”

“…However, this study also showed that deletion of the dsRBD did not significantly affect binding of the truncated protein to RNA . Our data and those of Wostenberg et al (2012) demonstrate that human Dicer dsRBD binds dsRNA with low micromolar or sub-micromolar affinity for RNA, hence less efficiently when compared to a dsRBD that has robust RNA-binding potential, e.g., dsRBD2 of XlrpA. Therefore, although Dicer's dsRBD likely contributes to binding of substrate RNA, its exact role in dsRNA processing still remains to be determined.…”

“…However, the difference between a 33-or 44-bp dsRNA and the siRNA-like 22-bp substrate was less dramatic than that revealed by our experiments. Additionally, the Dicer-dsRBD was also shown to be incapable of differentiating between pre-miRNAs and dsRNA substrates (Wostenberg et al 2012).…”

Engineering double-stranded RNA binding activity into the Drosha double-stranded RNA binding domain results in a loss of microRNA processing function

“…For the long ds44 and ds33 constructs, in vitro transcription using a duplex method, followed by purification, was performed as previously described [23]. The shorter ds22 RNA strands were purchased from Dharmacon and deprotected using the manufacturer protocol prior to duplex formation.…”

Characterization of a naturally occurring truncated Dicer

“…The PAZ/Platform, dsRBD and RNase III domains are involved in dsRNA binding and cleavage. In particular, PAZ and Platform domains enfold binding pockets for the 3 0 -overhang and 5 0 -phosphate moieties of the dsRNA substrate, respectively [3]; dsRBD domain binds the dsRNA [4]; RNase IIIa and IIIb domains function as an intramolecular pseudo-dimer forming a single processing centre containing two independent catalytic ''half sites'', with the RIIIa domain cleaving the 3 0 pre-miRNA arm, and RIIIb cleaving the 5 0 arm of pre-miRNA [5]. The helicase domain has a role in the control of enzyme activity [6] and interacts with the two dsRBD-containing cofactors of Dicer in RISC loading complex (RLC), i.e., TRBP and PACT, involved in siRNA-induced RNAi and in miRNA accumulation [7][8][9].…”