This characteristic ofalg-1loss of function pattern result in a retarded heterochronic phenotype observable by specific defect such as alae structure synthesis during the L4-adult transition stages (here represented by three lines). (C) Average and range of seam cells number at adulthood. The indicated strains were crossed with a strain expressing GFP in the seam cells (scm:: GFP) and the number of seam cells scored in young RS-1 adult. relationship with the miRISC at this developmental stage. The dispensability of AINs in miRNA regulation is further demonstrated by the capacity of ALG-1 tryptophan-binding pockets mutant to regulate a target of the embryonicmir-35microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins duringC. elegansembryogenesis. == Author Summary == Pet cells possess different small RNA species capable of precisely controlling the gene expression. Among them, microRNAs form a silencing complex with an Argonaute protein (known as miRISC) that abrogates protein production by targeting specific messenger RNAs. While there is a consensus that miRISCs are effective to mediate gene silencing, it is still unclear if they exist in different types in animals. Here we report specific mutations in theC. elegansmicroRNA-specific Argonaute ALG-1, which alter its relationship with the orthologs of GW182 proteins, important factors for miRISC-mediated silencing. Our genetic characterization of this mutant shows that part of miRISCs function without RS-1 the GW182 orthologs during the embryogenesis. These findings suggest the presence of unique miRISC that can regulate gene expression in different ways during animal development. == Intro == MiRNAs are highly conserved small non-coding RNAs that orchestrate gene expression in a broad range RS-1 of developmental processes. The production of miRNA implicates a successive two-step processing involving two RNase III enzymes, Drosha and Dicer, which cleave the primary and precursor miRNA molecules in the nucleus and cytoplasm, respectively. The 2123 nucleotide RNA products are RS-1 loaded onto Argonaute proteins to form the ribonucleoprotein complex referred to as microRNA induced silencing complex or miRISC (Reviewed in [1], [2]). Utilizing sequence complementarity, the miRNAs then guide the miRISC to the 3′ untranslated region (3’UTR) of target mRNAs to silence their expression. In humans andDrosophila, the miRISC is associated with a key partner protein, GW182, which contains glycine-tryptophan (GW) repeats. The GW182 N-terminal domain uses these GW repeats to interact with Argonaute [35], while the C-terminal domain recruits the PAN2-PAN3 and CCR4-CAF1-NOT deadenylase complex [68]. As a result, the complex triggers mRNA deadenylation and/or translational repression. Despite the difference in their domain organization from that ofDrosophilaand human GW182 proteins, two relatedC. elegansproteins, AIN-1 and AIN-2 [9, 10], appear to be orthologs of GW182 inC. elegans(reviewed in [11]). Both AIN-1 and AIN-2 are known to interact with Argonautes proteins through their GW repeats, but only AIN-1 interacts with PAN and never proteins [12] indicating that AIN-1 is most likely thebona fidefunctional GW182 ortholog. Thus, the interaction between Argonaute and GW182 proteins is clearly important for miRNA-mediated gene silencing across species, although the domain architectures of GW182 proteins are varied among species. Argonaute proteins have a bilobed structure, each composed of the N and PAZ domains or the MID and PIWI ones (Reviewed in [13]). The PAZ and MID domains are engaged in the guide-RNA recognition at the a few and 5 ends, respectively, while the PIWI domain harbors an RNase H-like active site that catalyzes the endonucleolytic cleavage of nucleic acids. Besides conferring the slicing activity on some Argonautes, the PIWI domain has also been reported to be important for the recruitment of silencing factors such as GW182 [4, 14, 15]. The crystal structure of human Argonaute2 (hAgo2) identified two hydrophobic pockets on the surface of the PIWI domain that were occupied with free tryptophan residues, suggesting that GW182 BTF2 proteins could be tethered to hAgo2 via these two pockets on the PIWI domain [16]. The physical interaction between hAgo2 and GW repeats was subsequently validated by NMR studies [17]. Furthermore, analogous binding pockets were also identified on the surface of human Argonaute 1 (hAgo1) [18], suggesting that these pockets could be a conserved feature for recruiting GW-proteins. Notably, although the molecular interactions between Argonaute and GW182 proteins have become clearer in recent years, the functional importance of this interaction in the context of.
