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Published on: September 25, 2013
GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans
Guillaume Jannot1,2, Pascale Michaud1,2, Miguel Quévillon Huberdeau1,2
1St-Patrick Research Group in Basic Oncology, CHU de Québec-Université Laval Research Centre (Hôtel-Dieu de Québec), Quebec City, Québec, Canada.
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.
Insights
The microRNA pathway can function without GW182 proteins during C. elegans development. This study shows that microRNA-induced silencing complex (miRISC) can operate independently of GW182 proteins in worm embryogenesis.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- MicroRNAs (miRNAs) and Argonaute proteins form the miRNA-induced silencing complex (miRISC).
- miRISC recruits GW182 proteins to mediate mRNA degradation and translational repression.
- The necessity of the miRISC-GW182 interaction for gene silencing in animal development remains unclear.
Purpose of the Study:
- To investigate the essentiality of the interaction between miRISC and GW182 proteins (AIN-1, AIN-2) for gene silencing during animal development.
- To determine if the microRNA pathway can function independently of GW182 proteins in *Caenorhabditis elegans*.
Main Methods:
- Utilized *Caenorhabditis elegans* as a model organism.
- Employed homology modeling to analyze Argonaute (ALG-1) structure and predict GW182 binding sites.
- Generated and analyzed mutations in ALG-1's tryptophan-binding pockets.
- Performed in vitro and in vivo experiments to assess protein interactions and gene silencing activity.
- Examined reporter gene silencing and phenotypic rescue in mutant animals.
Main Results:
- Homology modeling indicated conserved tryptophan-binding pockets in ALG-1 necessary for GW182 binding.
- Mutations in ALG-1's tryptophan-binding pockets disrupted in vitro and in vivo association with AIN-1 and AIN-2.
- ALG-1 mutants retained miRNA binding and processing but showed impaired reporter silencing.
- ALG-1 mutants phenocopied *alg-1* loss during larval stages but rescued embryonic lethality, suggesting dispensability of AINs at this stage.
- Mutant ALG-1 regulated a target of the embryonic mir-35 miRNA family, further supporting dispensability.
Conclusions:
- The microRNA pathway can operate independently of GW182 proteins during *C. elegans* embryogenesis.
- The interaction between miRISC and GW182 proteins is not essential for all developmental stages of miRNA-mediated gene silencing.
- This study reveals a developmental stage-specific dispensability of GW182 proteins in miRNA regulation.
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