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.
Highlights
The past decade has seen sustained interest in the role of small regulatory RNAs, most notably microRNAs and small interfering RNAs, in gene regulation
[1] Both small interfering RNA (siRNA) and miRNAs are processed by the RNase III enzyme Dicer into,21 nt RNAs prior to associating with Ago-family proteins, forming the RNA-induced silencing complex (RISC). [1,2] Human Dicer processes premiRNA more rapidly than pre-siRNA in the absence of cofactor proteins (e.g., TRBP and PACT), [9] demonstrating that Dicer is capable of discriminating between these two classes of substrate
The initial study was done with pre-mir-16-1, because it represents a dsRNA that Dicer would encounter in the cell and it correlates with previous work done with pri-mir-16-1 by our group and others. [23,46,47,48,49] Dicer-dsRNA binding domain (dsRBD) is able to bind pre-miRNA with a Kd = 2.260.1 mM (Figure 2 and Table 1), when fit to a general Hill equation binding model, as used in other binding studies. [23,50] Dicer-dsRBD binds pre-mir-16-1 more tightly than the first dsRBD of DGCR8 in isolation binds pri-mir-16-1 (Kd = 9.460.4 mM); Dicer-dsRBD binds pre-mir-16-1 slightly more tightly than DGCR8-Core, which contains two dsRBDs in tandem, does to pri-mir-16-1 (Kd = 3.760.1 mM). [23]
Summary
The past decade has seen sustained interest in the role of small regulatory RNAs, most notably microRNAs (miRNAs) and small interfering RNAs (siRNAs), in gene regulation Both of these RNA classes function in RNA interference (RNAi) by affecting gene translation through base-pairing with messenger RNA (mRNA) via their association with Argonaute-(Ago) family proteins. The PAZ domain of Dicer binds the 39 two-nucleotide overhang on the substrate – generated either by Drosha cleavage for miRNA or a prior round of Dicer cleavage for siRNA – and positions the catalytic sites of the intramolecular RNase III domain dimer for hydrolysis of each strand in the dsRNA, [12] resulting in cleavage to produce a new 39 two-nucleotide overhang. Drosophila Dicer-1 has been shown to engage the terminal loop of pre-miRNA with its helicase domain, establishing the correct distance from the PAZ domain for cleavage. [16] In a separate study, the helicase domains of both Drosophila Dicer-2 and of Caenorhabditis elegans Dicer-1 were shown to engage blunt-ended pre-siRNA models and establish a processive cleavage mode that is ATP-dependent. [17] These studies establish the importance of defining the functional roles of the peripheral domains found in metazoan Dicers
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