The Argonaute protein family
© BioMed Central Ltd 2008
Published: 26 February 2008
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© BioMed Central Ltd 2008
Published: 26 February 2008
Argonaute proteins were first discovered genetically, and extensive research in the past few years has revealed that members of the Argonaute protein family are key players in gene-silencing pathways guided by small RNAs. Small RNAs such as short interfering RNAs (siRNAs), microRNAs (miRNAs) or Piwi-interacting RNAs (piRNAs) are anchored into specific binding pockets and guide Argonaute proteins to target mRNA molecules for silencing or destruction. Various classes of small RNAs and Argonaute proteins are found in all higher eukaryotes and have important functions in processes as diverse as embryonic development, cell differentiation and transposon silencing. Argonaute proteins are evolutionarily conserved and can be phylogenetically subdivided into the Ago subfamily and the Piwi subfamily. Ago proteins are ubiquitously expressed and bind to siRNAs or miRNAs to guide post-transcriptional gene silencing either by destabilization of the mRNA or by translational repression. The expression of Piwi proteins is mostly restricted to the germ line and Piwi proteins associate with piRNAs to facilitate silencing of mobile genetic elements. Although various aspects of Argonaute function have been identified, many Argonaute proteins are still poorly characterized. Therefore, it is very likely that as yet unknown functions of the Argonaute protein family will be elucidated in the future.
Number of Argonaute family genes in different species
Number of genes
The sole Ago protein in S. pombe is involved in the establishment of heterochromatin and the silencing of transcription of specific genomic regions. It is therefore tempting to speculate that transcriptional silencing appeared earlier in evolution than post-transcriptional silencing processes. It has, however, been shown that the single S. pombe Ago can also guide post-transcriptional gene silencing when provided with an artificial reporter plasmid . Natural targets of post-transcriptional silencing in S. pombe have not yet been reported.
Additional structurally and functionally important motifs have recently been identified in Ago proteins (Figure 2b). Structural analysis of the sole Ago protein in the archaeon Archaeoglobus fulgidus revealed a third functionally important domain that resides between the PAZ and PIWI domains and is termed the MID domain. This domain binds the characteristic 5' phosphates of small RNAs and thus anchors small RNAs onto the Ago protein [22, 23]. Furthermore, the MID domain has been implicated in protein-protein interactions: Ago interactors such as Tas3 in S. pombe form a so-called 'Ago hook' that binds the MID domain of Ago proteins . Whether binding of protein interactors and small RNAs occurs simultaneously or is mutually exclusive remains unclear. Finally, Ago proteins contain a highly conserved motif similar to the 7-methylguanine (m7G) cap-binding motif of eukaryotic translational initiation factor 4E (eIF4E) . Two aromatic residues in human Ago2, Phe470 and Phe505, are required for this interaction. As some Ago proteins inhibit translational initiation of specific mRNA targets, it has been suggested that m7G-cap binding by Ago proteins may prevent eIF4E binding and therefore repress translation.
Drosophila germ cells are characterized by an electrondense, cytoplasmic structure called nuage, which is implicated in RNA interference (RNAi) and RNA processing and transport, and which contains the proteins Piwi and Aubergine (Aub), a member of the Piwi subfamily. Similar structures have been identified in mammalian germ cells and are called chromatoid bodies . Interestingly, chromatoid bodies contain members of the Piwi as well as the Ago subfamilies and might therefore be the germ-cell counterparts of somatic-cell P-bodies [33, 34].
Argonaute proteins have also been found in the nucleus. It is well established in plants that Ago4 directs siRNA-guided DNA methylation of chromatin [21, 35, 36]. Recently, it has been found that A. thaliana Ago4 (AtAgo4) localizes to distinct nuclear foci that are associated with the nucleolus and termed Cajal bodies. Cajal bodies are nuclear sites of ribonucleoprotein particle (RNP) assembly and it has been suggested that a larger DNA methylation complex containing AtAgo4, siRNAs and, presumably, many other factors is assembled in the Cajal bodies [37, 38]. In Drosophila, Piwi and Aub have been implicated in the silencing of transcription. Although it has not yet been shown directly, it is very likely that Piwi and Aub localize to the nucleus of Drosophila germ cells. In human cells, Ago1 and Ago2 have also been implicated in transcriptional silencing. siRNAs directed against promoters of different genes associate with Ago1 and Ago2 and silence gene expression at the transcriptional level [39, 40]. However, Ago proteins have not been clearly observed in mammalian cells by immunofluorescence studies, presumably due to the lack of highly sensitive antibodies.
Other miRNAs, on the other hand, predominantly bind to partially complementary target sites located in the 3' untranslated regions (UTRs) of their specific target mRNAs. Imperfect base pairing between small RNAs and their target mRNAs leads to repression of translation and/or deadenylation (removal of the poly(A) tail) of the target, followed by destabilization of the target, which most probably occurs in P-bodies (Figure 4) [47, 48]. The mechanism by which Ago proteins mediate translational repression is still a matter of debate. Ago proteins have been shown to act on translation initiation (summarized in ), on translation elongation [50–53] and on the degradation of nascent polypeptides . Therefore, the mechanisms by which Ago proteins inhibit translation might depend on the target that is being regulated. Such a model, however, remains to be experimentally proven.
It was shown very recently that upon cell-cycle arrest in human cells, Ago proteins bind to the 3' UTRs of specific mRNAs and stimulate translation. Interestingly, Ago proteins inhibit translation in proliferating cells and it has therefore been suggested that Ago-mediated translational regulation oscillates between repression and activation during the cell cycle [55, 56].
In Drosophila, zebrafish and mammals, miRNAs can guide deadenylation of target mRNAs [57–59]. In Drosophila, it became apparent that the interaction of Ago proteins with GW182, a protein required for miRNA function, is required for efficient deadenylation and subsequent degradation of target mRNAs by providing an interaction platform for the CCR:NOT complex, the major deadenylase complex in fly cells . Although members of the GW182 protein family are important for gene silencing in other organisms as well [26, 60–62], their mode of action remains elusive.
In S. pombe, small RNAs are transcribed from centromeric repeats and associate with Ago to form the RNA-induced initiation of transcriptional gene silencing (RITS) complex. RITS recruits methyltransferases to specific genomic regions, leading to methylation of Lys9 in histone H3 and the subsequent establishment of silenced heterochromatin (reviewed in ). In Drosophila, Piwi and Aub have been reported to be required for binding of the heterochromatin-specific protein HP1 to heterochromatic regions [64, 65]. Furthermore, Piwi, Aub and Ago3 (which belongs to the Drosophila Piwi subfamily) are required for transposon silencing in the Drosophila germ line [66–69]. Genetic studies revealed that Piwi is also required for germ-cell maintenance and it has recently been shown that Piwi has an additional role in epigenetic activation of distinct genomic loci . The functions of the Piwi subfamily members in mammals are largely unknown. In mice, the Piwi subfamily members are MIWI, MIWI2, and MILI. Male MILI and MIWI knockout mice are sterile, implicating a role for MILI and MIWI in spermatogenesis [71, 72]. MILI together with piRNAs has recently been shown to be involved in silencing of transposable elements in mouse testes [73, 74].
Although the Argonaute protein family was discovered only recently, many of their cellular functions are known and some have been characterized in detail. Nevertheless, many aspects of Argonaute function are still unclear. For example, it is intriguing that only human Ago2 shows slicer activity, even though the catalytic residues are conserved in other human Ago proteins. The answer to this question will probably have to await X-ray structures of human Ago proteins with their bound small RNAs, which have not yet been reported. The slicer activities of Argonaute proteins have not been analyzed in a wide range of organisms, and most of the natural slicer targets have not been found. Ago proteins are embedded in large regulatory networks  and future research will aim to describe and functionally characterize such networks. It will be very interesting to analyze how Ago regulatory networks are regulated themselves, as Ago proteins have been implicated in a variety of diseases, including fragile × syndrome , autoimmune diseases  and cancer . FMRp, the gene product affected in fragile × syndrome, has been found in biochemically purified Ago complexes and it has been suggested that FMRp is involved in miRNA biogenesis . Auto-immune antibodies directed against Ago proteins and other components of the RNAi pathway have been isolated from patients suffering from systemic rheumatic diseases. It is therefore tempting to speculate that larger Ago complexes are targeted by the immune system and that Ago proteins might contribute to the establishment of autoimmune diseases . Therefore, a detailed characterization of the biological roles of Ago proteins in conjunction with their associated small RNAs will not only help us to understand gene-silencing mechanisms but might also lead to a better understanding of numerous diseases.
We are grateful to M Beitzinger and L Weinmann for comments on the manuscript. Our research is supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft (DFG, Me2064/2-1), the Deutsche Krebshilfe and the European Union (LSHG-CT-2006-037900).