Michaud, Pascale et al. (2015) International Worm Meeting "A novel genetic approach to identify new factors implicated in Argonaute-dependent repression of mRNA."

Jannot, Guillaume, Michaud, Pascale, Simard, Martin J.

[International Worm Meeting, 2015]

MicroRNAs are short non-coding RNAs of 20 to 22 nt that repress the translation of target messenger RNAs. Once the mature miRNA is produced, it is loaded onto an Argonaute protein to form a silencing complex with the GW182 proteins (AIN-1 and AIN-2 in C. elegans). While recent observations indicate that cellular factors associated with the 3'UTR of mRNAs can actively contribute to miRNA-mediated gene silencing in animals, little is known about the identity of the genes implicated at this step of the pathway.We designed a novel genetic screen using a transgenic animal containing the gfp gene fused to the 3' UTR of cog-1 in which the natural miRNA lsy-6 binding sites were replaced by six box B sequences. This transgenic animal also expresses a functional alg-1 gene fused to a lambdaN peptide and tagged with mCherry. The lambdaN peptide specifically binds to the box B sequences and this tethering of ALG-1 to the 3' UTR of the gfp mRNA is sufficient to repress its expression. The repression of this reporter is therefore independent of miRNAs but still requires miRNA-specific Argonaute complex to fully block protein synthesis.A forward genetic screen was conducted to find mutants which misregulate the GFP transgenic reporter. The screen has allowed us to isolate candidates expressing both mCherry and GFP, suggesting the alteration of new genes necessary for the repression of the reporter. Characterization of these candidates is currently on going in order to identify the mutations responsible for this GFP derepression. With this innovative screen we will reveal genes specifically contributing with the miRNA-silencing complex to silence targeted mRNAs.

Frederick, Pierre-Marc et al. (2017) International Worm Meeting "Characterization of a new Argonaute interactor involved in the miRNA silencing pathway."

Jannot, Guillaume, Simard, Martin J., Frederick, Pierre-Marc, Villoing, Laure, Banville, Isabelle

[International Worm Meeting, 2017]

MicroRNAs are small non-coding RNA who regulates the expression of several genes. The miRNA pathway effector complex, the miRISC (miRNA induced silencing complex), is formed by the association of an argonaute protein with a miRNA. The subsequent binding of the miRISC to its mRNA target can either repress translation or degrade the mRNA. To further understand the miRNA gene regulation mechanisms, it is necessary to characterize new important molecular partners involved in the miRNA silencing. Using different genetic and molecular screens, we identified new miRISC interactors. Among them, we found a DnaJ chaperone protein as an interactor of a C. elegans miRNA-specific Argonaute ALG-1 in the yeast two-hybrid system. Different genetic assays indicate that the DnaJ chaperone is important for the microRNA pathway in animals. Interestingly, observations made with Drosophila cells extract suggested that Heat Shock Proteins or HSPs are required for small RNA loading onto Argonaute proteins (Iwasaki and al, Molecular Cell, 2010). It is also known that DnaJ chaperone proteins can provide specificity to HSPs in order to guide them to specific proteins. We therefore hypothesize that the DnaJ chaperone interacting with ALG-1 recruits HSPs proteins in order to permit loading of miRNAs on this Argonaute. To test this model, we will first demonstrate that DnaJ protein interacts with ALG-1 associated to the loading complex in vivo with the newly generated animals expressing endogenously HA-tagged DNaJ gene. We will next test whether DnaJ is required for: 1) the interaction of HSPs with ALG-1 and; 2) the loading of miRNA onto ALG-1 using both RNAi and KO animals. Taken together, this study will provide mechanistic insights on how microRNA (and potentially others small RNAs in worms) are loaded specifically onto the proper Argonaute proteins.