In mammalian cells, both microRNAs (miRNAs) and small interfering RNAs (siRNAs) are thought to be loaded into the same RNA-induced silencing complex (RISC), where they guide mRNA degradation or translation silencing, depending on the complementarity of the target. In Drosophila, Argonaute2 (AGO2) was identified as part of the RISC complex. AGO2 is an essential component for siRNA-directed RNA interference (RNAi) response and is required for the unwinding of siRNA duplex and in consequence, assembly of siRNA into RISC in Drosophila embryos. However, Drosophila embryos lacking AGO2, that are siRNA-directed RNAi-defective, are still capable of miRNA-directed target RNA cleavage. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, which is dispensable for siRNA-directed target RNA cleavage, is required for mature miRNA production that impacts on miRNA-directed RNA cleavage. The association of AGO1 with Dicer-1 and pre-miRNA also suggests that AGO1 is involved in miRNA biogenesis. These findings show that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism and suggest that there are inherent differences between siRNA-initiated RISCs and miRNA-initiated RISCs in Drosophila (Okamura, 2004).

The Drosophila RISC activity has been purified to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, homology was found between the PIWI domain of Ago-2; endonuclease V and potential active-site amino acid residues within the PIWI domain of Ago-2 were identified (Rand, 2004).

Double-stranded (ds) RNA induces the sequence-specific posttranscriptional gene silencing of cognate genes in numerous organisms. The multidomain ribonuclease III enzyme Dicer excises long dsRNA into duplexes of 21-23 nucleotides (nt), termed short interfering RNAs (siRNAs), which direct the cleavage of complementary mRNA targets, a process known as RNA interference (RNAi). Prior to target mRNA recognition, an siRNA duplex goes through an ATP-dependent unwinding process and one strand over the other is often preferentially loaded onto the RNA-induced silencing complex (RISC), the multiple-turnover enzyme complex that mediates endonucleolytic cleavage in the RNAi pathway. The RISC is guided to cleave target mRNAs sharing perfect complementarity across the center of the complementary siRNA strand in the absence of high-energy cofactors. siRNAs are not the only products of Dicer. Natural dsRNA-encoding genes, named microRNA (miRNA) genes, encode RNA products of ~70 nt that are predicted to form imperfect hairpin structures and are processed by Dicer to mature 21-23-nt miRNAs. Only one Dicer enzyme is found in C. elegans and humans, therefore indicating that the same Dicer is required for both RNAi and for the processing of miRNA precursors in these organisms. The expression of miRNAs is often developmentally regulated, suggesting an important role for miRNAs in the regulation of endogenous gene expression. Target mRNAs containing sequences imperfectly complementary to the miRNA can be subject to translational repression without altering mRNA stability (Okamura, 2004 and references therein).

Recent findings point to a tight connection between miRNA and RNAi molecular machineries. Both miRNAs and siRNAs have been shown to be capable of target mRNA degradation or translation silencing in mammalian cells and plants. These findings imply that, regardless of the maturation process, once the small RNA is loaded, the RISC uses it to degrade or inhibit translation depending on the degree of complementarity between the small RNA and its mRNA target (Okamura, 2004 and references therein).

In C. elegans, genetic analyses suggest that RDE-1, a member of the Argonaute family of proteins, is required for the initiation of RNAi with injected dsRNAs, whereas Alg-1 and Alg-2, other Argonaute family members, are required for the accumulation of stable mature miRNAs in C. elegans, but not for RNAi driven by dsRNA. These results suggest that distinct members of the Argonaute family of proteins in C. elegans may provide specificity to their respective pathways, RNAi and translation inhibition. However, the underlying molecular mechanisms are not well understood (Okamura, 2004).

In Drosophila, it has been shown that Argonaute2 (AGO2) protein, a member of the Argonaute family of proteins, is essential for RNAi driven by exogenously introduced dsRNA and is the first protein component to be identified as part of the RISC complex in cultured Drosophila S2 cells (Hammond, 2001). Although AGO2 is known to associate with several proteins such as the Drosophila homolog (dFMR1) of the human fragile X mental retardation protein and Vasa intronic gene (Vig) (Caudy; 2002; Ishizuka; 2002), the precise role that AGO2 plays in RNAi is not well understood. In this paper, AGO2 deletion mutant flies have been produced, and it has been found that embryos lacking AGO2 are siRNA-directed RNAi defective but are still capable of miRNA-directed target RNA cleavage. AGO2 mutant embryos are impaired in the assembly of siRNA into RISC. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, is dispensable for siRNA-directed RNA cleavage but is necessary for the accumulation of stable mature miRNAs, and thus impacts on miRNA-directed target RNA cleavage. These findings suggest that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism, and provide specificity to their respective pathways in Drosophila (Okamura, 2004).

In mammals, there appear to be no real distinctions between the siRNA and miRNA pathways downstream of Dicer. In contrast, genetic studies in C. elegans have shown that distinct Argonaute homologs appear to be dedicated to distinct RNAi and translation repression pathways. Using target RNA cleavage assays, this study shows that siRNA-directed RNA cleavage depends on AGO2 but does not require AGO1, whereas miRNA-directed RNA cleavage depends on AGO1 but does not need AGO2 in Drosophila embryos. These results suggest that maturation and the function of siRNAs and miRNAs have differential requirements for Argonaute proteins in Drosophila. These findings also suggest that Argonaute proteins regulate entry points of small RNAs to RISC but may not act as determinants for target RNA cleavage or translation repression. AGO2 is an essential component of RNAi and part of the RISC complex (Hammond, 2001). The particular function of AGO2 lies downstream of siRNA duplex production in the RNAi pathway. It has been shown that duplex siRNAs are incorporated in RISC precursors and ATPdependent unwinding of siRNAs converts RISC precursors into active RISC that then degrades the specific target mRNAs. Thus, AGO2 is thought to function at some or all of these steps in RNAi. Both the unwinding of siRNA duplex and the assembly of siRNA into functional RISC are impaired in AGO2 mutant embryos. Therefore, these results indicate that AGO2 functions at a step(s) in RISC assembly after binding of the siRNA duplex to RISC precursors. Recently, Liu (2003) demonstrated that Dicer-2 not only associates with siRNA production, but also, together with R2D2, facilitates siRNA loading onto RISC in Drosophila. These data suggest that AGO2, together with Dicer-2 and R2D2, assembles siRNA into functional RISC in Drosophila embryos (Okamura, 2004).

AGO2⁴¹⁴ flies are developmentally normal, which provides circumstantial evidence that AGO2 is not important for development and by inference, not essential for utilization and function of miRNA that often regulates the developmental pathways. In contrast, AGO1 is required for the stable production of mature miRNAs; this might explain the fact that AGO1 is essential for normal development, particularly in the nervous system. The physical association of AGO1 with Dicer-1 and premiRNA suggests that AGO1 is involved in miRNA biogenesis. In fact, recent genetic studies of Dicers in Drosophila have shown that mutations in Dicer-1 block processing of miRNA precursors (Lee, 2004). Together, these results suggest that in Drosophila, distinct pathways exist for siRNA and miRNA production and their concomitant assembly into RISC complexes. Do AGO1 and AGO2 functional specificities reflect physically distinct miRNA-associated RISC and siRNA-associated RISC, or differential processing and/or loading of small RNAs into generic RISC? Although there is no definitive evidence that there is a generic RISC for both small RNAs, recent studies have shown that the RISC containing active miRNAs and the RISC involved in siRNAdirected RNAi are very similar, if not identical, since endogenous miRNAs can cleave mRNAs with perfect complementarity, and exogenously introduced siRNAs can translationally repress mRNAs bearing imperfectly complementary binding sites. The fbindings show that the miRNA-associated RISC that cleaves RNA does not need AGO2, whereas siRNA-associated RISC does. This argues that there are inherent differences between siRNA-initiated RISC and miRNA-initiated RISC in Drosophila. Physically, they might be the same generic complex (for instance, both containing AGO2) but one does not need AGO2 activity. It is also possible that they might be physically distinct with respect to Argonaute proteins (Okamura, 2004).

AGO1 is not present in AGO2-associated complexes and can be biochemically separated from siRNA-loaded RISCs (Caudy, 2002). Therefore, AGO1 and AGO2, conceivably, are not incorporated into the same RISC. However, miRNAs and siRNAs are found, to some extent, in both AGO1 and AGO2 complexes, suggesting that association of Argonaute proteins with small RNAs is preferential rather than absolutely specific. Alternatively, a fraction of small RNAs might be exchangeable between the two complexes, probably during recycling of their silencing function. A recent study has shown that miRNA-associated RISCs and siRNA-associated RISC are different with respect to Dicers in Drosophila (Lee 2004). The current results clearly suggest that in conjunction with Dicers, Argonaute proteins regulate the formation of siRNA-associated RISC or miRNA-associated RISC, since their particular functions lie downstream of Dicers in a step(s) in the assembly of small RNAs into RISC (Okamura, 2004).

GENE STRUCTURE

cDNA clone length - 4050

Bases in 5' UTR - 90

Exons - 9

Bases in 3' UTR - 315

PROTEIN STRUCTURE

Amino Acids - 694

Structural Domains

RNA interference is a conserved mechanism that regulates gene expression in response to the presence of double-stranded (ds)RNAs. The RNase III-like enzyme Dicer first cleaves dsRNA into 21-23-nucleotide small interfering RNAs (siRNAs). In the effector step, the multimeric RNA-induced silencing complex (RISC) identifies messenger RNAs homologous to the siRNAs and promotes their degradation. The Argonaute 2 protein is a critical component of RISC. Both Argonaute and Dicer family proteins contain a common PAZ domain whose function is unknown. The three-dimensional nuclear magnetic resonance structure of the Drosophila Ago2 PAZ domain has been examined. This domain adopts a nucleic-acid-binding fold that is stabilized by conserved hydrophobic residues. The nucleic-acid-binding patch is located in a cleft between the surface of a central beta-barrel and a conserved module comprising strands beta3, beta4 and helix alpha3. Because critical structural residues and the binding surface are conserved, it is suggested that PAZ domains in all members of the Argonaute and Dicer families adopt a similar fold with nucleic-acid binding function, and that this plays an important part in gene silencing (Lingel, 2003).

The solution structures of the Argonaute2 PAZ domain bound to RNA and DNA oligonucleotides have been examined. The structures reveal a unique mode of single-stranded nucleic acid binding in which the two 3'-terminal nucleotides are buried in a hydrophobic cleft. It is proposed that the PAZ domain contributes to the specific recognition of siRNAs by providing a binding pocket for their characteristic two-nucleotide 3' overhangs (Lingel, 2004).

RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. The crystal structure of the PAZ domain of Drosophila Argonaute2 has been solved. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway (Song, 2003).

Purification of RISC activity suggested that Ago-2 must have an unidentified nuclease activity. A bioinformatics approach was taken to help find the region that is responsible for this putative activity. Similarity search engines based purely on sequence (e.g., position-specific-iterative BLAST) have been unable to detect any homology between Ago-2 and known nucleases. Instead, an algorithm that identifies relationships based on a combination of sequence profiles with secondary-structure prediction, METABASIC, was used. Found homology should imply similarity in fold and possibly function. The PIWI domain of Drosophila Ago-2 found the endonuclease V family as the first METABASIC hit with the score of 15.2. METABASIC scores of >12 correspond to <5% incorrect matches. Unfortunately, no mutants or structures from the endonuclease V protein family have been reported that allow for identification of the active site. However, a region of amino acid conservation between the endonuclease V family and the better characterized UVRC DNA-repair endonuclease families active site was used to predict where the endonuclease V active site should be. In turn, this alignment allowed a prediction to be made of the residues that might form the active site of PIWI based on obvious conservation between the active sites. Sites aspartate 965 (GADVT), glutamate 1016 (TLEHL), and aspartate 1037 (YRDGV) of Drosophila Ago-2 are predicted as likely active-site residues for the Ago-2 nuclease activity. Coordination by these three residues with water coordinated in position to make the attack on the phosphodiester backbone would account for the normal tetrahedral coordination of a Mg2+-dependent nuclease (Rand, 2004).

EVOLUTIONARY HOMOLOGS

For information of Argonaute 2 homologs see Argonaute 1 site.

date revised: 7 January 2005

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.