Abstract
MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1. Homology modeling based on human Argonaute structures indicated that ALG-1 possesses conserved Tryptophan-binding Pockets required for GW182 binding. We show in vitro and in vivo that their mutations severely altered the association with AIN-1 and AIN-2. ALG-1 tryptophan-binding pockets mutant animals retained microRNA-binding and processing ability, but were deficient in reporter silencing activity. Interestingly, the ALG-1 tryptophan-binding pockets mutant phenocopied the loss of alg-1 in worms during larval stages, yet was sufficient to rescue embryonic lethality, indicating the dispensability of AINs association 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 embryonic mir-35 microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins during C. elegans embryogenesis.
Highlights
MiRNAs are highly conserved small non-coding RNAs that orchestrate gene expression in a broad range of developmental processes
MiRISC Silencing without GW182 in C. elegans Embryos (PRESTO) from the Japan Science and Technology (JST) Agency
Despite the difference in their domain organization from that of Drosophila and human GW182 proteins, two related C. elegans proteins, AIN-1 and AIN-2 [9, 10], appear to be orthologs of GW182 in C. elegans. 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 NOT proteins [12] indicating that AIN-1 is most likely the bona fide functional GW182 ortholog
Summary
MiRNAs are highly conserved small non-coding RNAs that orchestrate gene expression in a broad range of developmental processes. Despite the difference in their domain organization from that of Drosophila and human GW182 proteins, two related C. elegans proteins, AIN-1 and AIN-2 [9, 10], appear to be orthologs of GW182 in C. 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 NOT proteins [12] indicating that AIN-1 is most likely the bona fide functional GW182 ortholog. The interaction between Argonaute and GW182 proteins is clearly important for miRNA-mediated gene silencing across species, the domain architectures of GW182 proteins are varied among species
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