RAB protein controls Argonaute localization for proper miRNA function in C. elegans

MicroRNAs are critical regulators of gene expression in animals. To repress their target mRNAs, these small RNAs are first loaded onto Argonaute proteins to form the silencing complex called miRISC. The complex must then be transported to its target mRNA where it interacts mostly within the 3’UTR through base-pairing. After target repression, the constituents of the miRISC undergo degradation or recycling mediated through their accurate sorting and localization in the cell. While some reports have linked intracellular trafficking to miRNA activity, it is still unclear how these pathways coordinate to allow proper miRNA-mediated gene silencing and turnover. Through a forward genetic screen performed in the nematode Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the miRNA pathway. We show that TBC-11 is likely a GAP for the small GTPase RAB-6 and that its regulation is required for miRNA function in animals. The absence of functional TBC-11 leads to the persistent activation of RAB-6, which increases the localization of miRNA-unloaded Argonaute ALG-1 on endomembranes. This inactive form of Argonaute accumulates on polysomes, leading to defective miRNA-mediated target mRNA repression. Together, our results establish the importance of intracellular trafficking for miRNA function and demonstrates the involvement of a small GTPase in proper Argonaute localization in vivo. Develop a novel protocol to clone small RNA and mRNA