The biochemical study of ALG-1 and ALG-2 confirms that these Argonautes have retained the molecular characteristics found in catalytically competent Argonautes of Schizosaccharomyces pombe
, plants and other animals. The enzymatic activity of ALG-1/2 requires both magnesium (a divalent cation) (16
) and the DDH motif within the PIWI domain of the proteins (37
). We also observed that recombinant ALG-1/2 can bind small RNA duplexes and is capable of separating strands from miRNA-like molecules that contain mismatches. Similar data have been reported for human Argonautes (30
). Recent studies performed with Drosophila
embryo lysates demonstrated that the miRNA-specific Argonaute Ago1 protein also displays a preference for imperfectly paired duplexes and can only form functional miRISC complexes once loaded with these types of RNA molecules (39
). In all these conditions, it is possible that the presence of a complementary target RNA to the guide strand contributes to the release of the passenger strand by Argonautes. Therefore, our results support the idea that miRNA-specific animal Argonautes have the capacity to release the miRNA passenger strand from a Dicer-processed miRNA duplex to form the functional miRISC.
The first study of C. elegans alg-1
Argonautes by Grishok et al.
demonstrated that the loss of both Argonautes led to a decrease of mature let-7 and lin-4 miRNAs and an accumulation of pre-lin-4 molecules (20
). Although this initial observation suggested a role for ALG-1 and ALG-2 in the stability and production of miRNAs in C. elegans
, it was still unclear how these proteins could contribute to miRNA biogenesis. The data presented herein reveal the role of ALG-1 and ALG-2 in miRNA production and demonstrate, for the first time, the importance of ALG-1/2 slicing activity in processing multiple miRNAs. Furthermore, our studies unveil the functional differences of these miRNA-specific Argonautes in processing different miRNA species. Although the replacement of either ALG-1 or ALG-2 with their respective catalytically inactive forms had no observable effect in animals, the loss of slicing activity for both miRNA-specific Argonautes led to severe developmental defects that are associated with the loss of miRNAs in C. elegans
). At the molecular level, we observed that the loss of this enzymatic activity in miRNA-specific Argonautes led to the accumulation of mature miRNAs along with the appearance of a shorter form of miRNA precursor molecules. These observations suggest that in the absence of slicing-competent miRNA-specific Argonautes, mature miRNAs remain associated with the complementary strand found in precursor miRNA molecules, leading to a significant decrease of miRISC that is essential for animal viability. In addition, our data also support the idea that Dicer can cleave independently RNA strands from pre-miRNA substrates.
Several lines of evidence in our data support the idea that Dicer can cleave only one strand of the precursor miRNA: (i) the size of the aberrant precursor miRNA form is about 21 nt shorter than the pre-miRNA and (ii) the mature form for all miRNA tested was detectable by northern blot. Although many in vitro
and in vivo
studies demonstrate that the Dicer enzyme can cleave perfectly paired dsRNA on both strands (40–44
), some experimental evidence also supports that this enzyme can separately cleave both strands of a miRNA precursor. In vitro
analysis of Drosophila
Dcr-1, which is required for the miRNA pathway, demonstrated that a single mutation in the active site of one of RNase III domains produced a 21-nt miRNA and a truncated miRNA precursor (45
). Recently, it has been shown that cells carrying specific point mutations within each RNAse III domain of human Dicer display defects in the production of either 5p or 3p miRNAs (46
). Biochemical studies on recombinant human Dicer also demonstrate its enzymatic capacity to produce a truncated miRNA precursor and a mature miRNA in vitro
). In agreement with those observations, our data indicate that at least one Argonaute competent slicer is essential to enable complete Dicer cleavage of pre-miRNAs. Therefore, in absence of the slicing Argonautes, Dicer may cleave only once and thus affects the formation of miRISC. Alternatively, we can also consider that after Dicer produces a single 21-nt molecule from the pre-miRNA, slicing Argonautes may be important to cleave within the complementary sequence to trigger the degradation of the truncated precursor.
Previous observations indicated that Argonaute proteins and Dicer are found in the same complex and form the RISC loading complex (50–54
). It has recently been observed that human Ago2 can bind some pre-miRNA molecules even in absence of Dicer (55
). Therefore, the presence of co-factors such as slicing competent Argonautes might be essential to coordinate Dicer cleavage in animals. Future analysis with purified RISC loading complex will help in gaining insight about the contribution of slicing Argonaute proteins to this process.