Sandbox Reserved 1106

The AGO1 protein is part of the Argonaute family of proteins [Argonaute]. It is ubiquitously expressed in all higher eukaryotes. Similarly to other proteins of the family, it intervenes in the function of RNA interference through the binding of siRNA and the forming of RISC complexes. Unlike other Argonaute proteins, it can act by itself in the case of a “minimal RISC” by blocking instead of destroying the mRNA. For other proteins of the family, see Argonaute.

Structure

Length and domains

For more details on the 3D structure domains, see the page Argonaute 3D structures in the rubric Argonaute 1

Primary Structure

The Ago1 protein has the same 4 primary domains as all argonaute proteins (N, PAZ, Mid, PIWI, described in the page Argonaute) and the two linker regions L1 (also called DUF1785 domain) and L2. In fact, there are 84% of similitudes between the primary sequence of hAgo1 and that of hAgo2. The main structural difference between the two similar proteins is that the N domain of AGO1 interacts with the L1, L2 and PIWI domains via residues 18-48 and 138-173.

Besides, Argonaute 1 can be found in a lot of phylogenetic groups, with different structure. For example, in the plant Brachypodium distachyon, where 10 argonautes proteins have been discovered, BdAGO1 lacks the N and Mid domains. This particularity explains the small size of this protein with only 624 residues. Moreover, BdAGO1 is functionally related to Ago1 as both proteins lack endonuclease activity. Indeed, the catalytic tetrad of its PIWI domain is disabled through the absence of the last D/H residue in the domain.

However, the AtAGO1 (from Arabidopsis thaliana), which can be found in the nucleus and the cytoplasm of the plant, includes a Mid domain and has an homologous conformation to BdAO9, BdAGO11, BdAGO12, BdAGO15, and BdAGO16. On the other hand, BdAGO1 has AtAGO4 as its closest homolog (like BaAGO2, BdAGO3 and BdAGO4), even if the AtAGO4 possess a Mid Domain. ^[1]

Proteins Associating with Ago1

The Argonaute 1 protein can be associated with others molecular components of the cell. In fact, proteomic analysis has demonstrated that RNase III Dicer binds hAgo1 through the PIWI domain (see page on argonaute) and also, at least, four other proteins in specific association with hAgo1:

• TNRC6B isoform 1 / KIAA1093 (175kDa) which has RRM motif at C-term to recognize RNA, and also GW repeats (trinucleotide motifs)

• MOV10 (130kDa)

• PRMT5 (arginine methyl-transferase) (70kDa)

• Additionally, the translation factor eEF1α (50kDa) is thought to associate with AGO1

hAgo1 in a complex with let-7

The hAgo1 interact with an endogenous let-7 in a similar manner to the manner in which hAgo2 interact with miR20a, but with some slight differences. From a domain point of view, the position of L1 linker is the same for hAgo1/let-7 and hAgo2/miR20a. Also, the N subdomain (from residues K49 to S137 of the N domain) interacts with the endogenous sequence in a loosely similar way for both hAgo1 and hAgo2 except a 3A translation toward the PIWI domain in hAgo1. Besides the piece of information gets from the N subdomain may suggest an interaction with the guide-target duplexes. On the other hand, the PAZ domain is folded differently in hAgo1/let-7 in order to be placed away from the PIWI domain.

Then, from a nucleotide point of view, hAgo1 shows 6 RNA-specific interactions with ribose 2’OH of let-7 (U1, G2, G4, G5, A7 and A8). There are either direct or water-mediated through the side- or main- chain atoms of hAgo1 like for hAgo2/miR20a. However, there is one interaction presents in hAgo2/miR20a that is missing in hAgo1/let-7 ; it is the one with U6 since this base is slightly modified because of the shift of α7 in the L2 toward the N domain. Furthermore, the first adenine base has a syn conformation around the glycosidic bond and the last two bases are piled in the PAZ domain

Finally, one of the most important aspect is the non-5’ phosphorylated strand ; indeed the U1 base of let-7 and the 5’P of hAgo1 are interacted with protein residues of the Mid and PIWI domains which avoid an interaction between them, preventing a phosphorylation which is important for accurate slicing activity.

Knowing that the nine first 9 nucleotids of let-7 are: 5’AAUAUUAAA3’.

GW motifs

The hAGO1 protein has a single GW binding site. This site is able to bind to GW/WG motifs of other proteins, such as GW182. It is contained in the PIWI domain, also found in AGO2. Proteins like GW182 form with their GW motif a “hook” which interacts with the PIWI domain and enables the docking of the protein in a tryptophan-binding pocket of AGO1. This interaction is enhanced through the binding of an miRNA to AGO1, which ensures that mature RISC can be recruited efficiently for silencing. Through this kind of interaction, several AGO1 proteins can bind to a single protein with GW motifs.

DDB2 and Ago 1 complex

In the case of UV-irradiation response, it has been shown that AGO1 can form complexes with DNA Damage Binding (DDB) proteins and uviRNAs. The specific mechanisms of binding and binding sites have not yet been uncovered.

Mutations domains

Restoration of slicing activity

Among the four human Argonaute proteins, only hAGO2 is an active slicer. A conserved catalytic triad within the PIWI domain in hAGO2 is required for its slicing activity and hAGO1 has an arginine in place of the active site histidine in the DEDH tetrad. Yet, restoring an intact catalytic DEDH tetrad with a R805H mutation is not enough to activate slicing in hAGO1. Therefore, it appears more distant regions of the enzyme are determinant for slicer activity. Domain-swapping experiments revealed that the substitution of the hAGO1 and hAGO2 PIWI domain activates hAGO1 while hAGO2 slicing activity is being removed; providing new evidence that other factors, in addition to the incomplete DEDH tetrad, are responsible for the slicer defect in hAGO1. Indeed, an additional mutation of L674F on the PIWI loop 3 adjacent to the active site of hAGO1 leads to an active slicer with level comparable to that of the hAGO2 PIWI domain swap. Intriguingly, the elements that make hAGO1 an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme.

Function

Non proteolytic activity

Unlike hAGO2, hAGO1 does not function as an endonuclease that can cleave mRNA molecules within regions that base pair with perfectly complementary siRNAs or miRNAs. The mechanism by which hAGO1 mediates translational repression is still a matter of debate. hAGO1 has been shown to act on translation initiation, on translation elongation and on the degradation of nascent polypeptides. Therefore, the mechanisms by which hAGO1 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, hAGO1 bind to the 3’ UTRs of specific mRNAs and stimulate translation. Interestingly, hAGO1 proteins inhibit translation in proliferating cells and it has therefore been suggested that hAGO1-mediated translational regulation oscillates between repression and activation during the cell cycle.

Role in RISC complex

The AGO1 protein is part of the RNA-induced silencing complex RISC, which is required in the RNA interference process. In this case, the miRNAs, interacting with hAGO1, bind to partially complementary target sites located in the 3’ unstranslated 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.

Applications

Cancer Research

Studies have linked a reduced amount of Argonaute proteins in cells with an increase in tumour development and progression in the case of melanoma. As important parts of miRNA processing, several key cellular processes linked to gene silencing are impacted by the amounts of AGO proteins in the cell. Additionally, although there is few research in the case of AGO1, another Argonaute protein, AGO2, was found to be overexpressed in carcinomas and to be interacting with well-studied tumour factors in regulating the metabolism of the cancer cells