Purified Dicer-1 and Dicer-2 both process pre-miRNAs, but generate different length products (22 versus 21 nt). Genetic analyses suggest that Dicer-1 and Dicer-2 are restricted to specific substrate classes in vivo (Lee et al. 2004
). For example, Dicer-2 cannot replace Dicer-1 in the miRNA pathway. Similarly, dicer-2
mutants are defective for RNAi, even though they express normal levels of Dicer-1 (Lee et al., 2004
). Despite structural similarities, Dicer-2 specifically processes esiRNA hairpins, while Dicer-1 cleaves pre-miRNAs (Lee et al., 2004
; Förstemann et al., 2005
; Jiang et al., 2005
; Saito et al., 2005
; Miyoshi et al., 2010
). This observation suggests that the length of a dsRNA is the primary determinant of substrate choice.
Our data argue that the combination of R2D2 and cellular phosphate restricts Dicer-2 to its biologically relevant substrates by inhibiting the processing of short substrates such as pre-miRNA. Thus, a protein, R2D2, and a small molecule, phosphate, convert a promiscuous dsRNA endonuclease into one specific for the long dsRNA substrates that trigger RNAi. It is tempting to speculate that inorganic phosphate interferes with recognition of the 5′ monophosphate present on all pre-miRNAs, and that 5′ phosphate recognition is unnecessary for longer substrates, because their greater length allows additional protein-RNA contacts—perhaps by the dsRNA-binding and the helicase domains—between Dicer-2 and long dsRNA. We note that human Dicer has been reported to recognize a 5′ monophosphate on single-stranded RNA (Kini and Walton, 2007
In flies, the Dicer-2 partner proteins Loqs-PD and R2D2 likely enhance substrate specificity by increasing the affinity of the enzyme for long dsRNA. The kcat
for and Dicer-2 and Dicer-2/R2D2 were similar, but R2D2 decreased the KM
of Dicer-2 for long dsRNA ( and Figure S5
). We note that the specificity constant, kcat
, was ~4 fold higher for the Dicer-2/R2D2 heterodimer than for Dicer-2 alone. Similarly, Loqs-PD lowered the KM
of Dicer-2 for long dsRNA without reducing the catalytic rate, resulting in a ~10-fold higher kcat
Processing of pre-miRNA by Dicer-1 was unaffected by phosphate. We find that the intrinsic properties of Dicer-1, which cannot efficiently catalyze multiple-turnover processing of long dsRNA, restrict that enzyme to process pre-miRNA. We do not yet know whether the transition of substrates from Dicer-1 to Dicer-2 is gradual, such that some substrates are processed equally well by both enzymes. In theory, such intermediate substrates might be selected against in evolution, enforcing the distinction between Dicer-1 and Dicer-2 substrates.
The Dicer-2 helicase domain is similar to that of RIG-I, a sensor in the mammalian innate immune system. The RIG-like ATPase/helicase domain is conserved among plant and animal Dicers. Yet, its function has remained unknown. Our data suggest that this domain of Dicer-2 is involved in ATP-dependent production of successive siRNAs from long dsRNA. Notably, two other members of this helicase family, DRH-3 and RIG-I are also bona fide ATPases: DRH-3, a C. elegans
protein required for RNA silencing and germ-line development (Nakamura et al., 2007
), is a dsRNA-stimulated ATPase (Matranga and Pyle, 2010
); and the mammalian protein RIG-I, which recognizes viral 5′ triphosphorylated dsRNA and initiates an innate immune response, uses ATP to translocate along dsRNA (Myong et al., 2009
). Our data are consistent with the idea that ATP hydrolysis fuels translocation of Dicer-2 along long dsRNA substrates. An alternative view—that the ATP-dependent binding of a molecule of Dicer at the end of the substrate promotes the complete and rapid oligomerization of Dicer-2 along the entire extent of the dsRNA—would require that the Dicer and RIG-I helicase domains share a conserved sequence but have highly divergent functions.
ATP was not required for Dicer-2 to process pre-miRNA, and a mutant Dicer-2 unable to hydrolyze ATP remained able to process pre-miRNA but not long dsRNA. These results help explain why in C. elegans
, in which a single Dicer processes both long dsRNA and pre-miRNA, a mutation in the DCR-1 helicase domain disrupted endo-siRNA, but not miRNA, accumulation (Welker et al., 2010
Four lines of evidence support a role for ATP hydrolysis in the production of successive siRNAs along the dsRNA by Dicer-2. First, Dicer-2 consumes a constant amount of ATP per base-pair. Second, ~23 molecules of ATP were consumed for each 21 nt siRNA produced. Third, the rate of production of the first, second, and fourth siRNAs from a long dsRNA substrate were indistinguishable in the presence of ATP, but in the absence of ATP, the rate of siRNA production declined with increasing distance from the end of the dsRNA. Finally, the association of Dicer-2 with a long dsRNA was resistant to dilution provided ATP was present, suggesting that after binding the end of its substrate, Dicer-2 remains bound to the dsRNA and uses ATP energy to reposition itself to produce the next 21 bp siRNA. Translocation along the dsRNA seems a likely mechanism.
Although helicase mutant Dicer-2G31R
processed pre-miRNAs as efficiently as wild-type Dicer-2, the mutant was unable to produce even the terminal siRNA from a long RNA duplex under multiple turnover conditions. This suggests an inherent ability of the helicase domain of Dicer-2 to distinguish between long and short substrates. We note that the helicase domain of human Dicer auto-inhibits processing of an RNA duplex, and its dsRNA-binding protein partner TRBP, a homolog of R2D2 and Loqs, relieves this inhibition (Ma et al., 2008
; Chakravarthy et al., 2010
). We hypothesize that Drosophila
Dicer-2 can occupy two distinct conformations. When inorganic phosphate is low, Dicer-2 assumes a conformation—perhaps similar to the auto-inhibited conformation of human Dicer—that can bind and load siRNA. This conformation is unaffected by ATP and, we presume, is involved in promiscuously processing pre-miRNA in vitro. When inorganic phosphate is higher and the enzyme’s helicase and dsRNA-binding domains engage its substrate, Dicer-2 assumes a conformation that requires ATP for binding and hydrolysis to process dsRNA.