A New Role for the GARP Complex in MicroRNA-Mediated Gene Regulation

Abstract

Many core components of the microRNA pathway have been elucidated and knowledge of their mechanisms of action actively progresses. In contrast, factors with modulatory roles on the pathway are just starting to become known and understood. Using a genetic screen in Caenorhabditis elegans, we identify a component of the GARP (Golgi Associated Retrograde Protein) complex, vps-52, as a novel genetic interactor of the microRNA pathway. The loss of vps-52 in distinct sensitized genetic backgrounds induces the enhancement of defective microRNA-mediated gene silencing. It synergizes with the core microRNA components, alg-1 Argonaute and ain-1 (GW182), in enhancing seam cell defects and exacerbates the gene silencing defects of the let-7 family and lsy-6 microRNAs in the regulation of seam cell, vulva and ASEL neuron development. Underpinning the observed genetic interactions, we found that VPS-52 impinges on the abundance of the GW182 proteins as well as the levels of microRNAs including the let-7 family. Altogether, we demonstrate that GARP complex fulfills a positive modulatory role on microRNA function and postulate that acting through GARP, vps-52 participates in a membrane-related process of the microRNA pathway.

Farnesylated heat shock protein 40 is a component of membrane-bound RISC in Arabidopsis

“…For example, mutants in the Arabidopsis HSP90 co-chaperone SQN show weak miRNA-related defects on their own, but the importance of SQN for miRNA activity is revealed by its spectacular genetic interaction with weak ago1 mutant alleles ( 8 ). Similarly, at low temperature, mutants in the C. elegans AGO protein ALG-1 show weakly penetrant defects in developmental transitions controlled by the lin-4 and let-7 miRNAs, but those phenotypes become strongly exacerbated upon mutation of components of the Golgi-associated retrograde protein complex that affects miRNA levels, including those of the let-7 family ( 43 ). To test for such synthetic interactions, we constructed two sets of double mutants with era1–2 : the first with a hypomorphic mutant allele of the key miRNA biogenesis factor DICER-LIKE1 ( dcl1–11 ( 44 , 45 )), and the second with the hypomorphic ago1–27 allele ( 46 ).…”

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for micro RNA ‐guided gene silencing in vivo

“…Seam cell fate throughout development is regulated by the miRNAs lin-4 (Ambros & Horvitz, 1984) and the let-7 family (miR-241, miR-84, miR-48, and let-7;Reinhart et al, 2000;Abbott et al, 2005). In animals depleted of alg-1, the L2 proliferative division is reiterated in subsequent larval stages, resulting in an excess number of seam cells (Grishok et al, 2001;Vasquez-Rifo et al, 2013). As expected, S992A and S992E mutants also showed an abnormal number of seam cells ( Fig 5C) as well as a misregulation of two let-7 family targets hbl-1 and daf-12 implicated in the control of proliferative seam cell division (Abbott et al, 2005;Hammell et al, 2009) (Appendix Fig S3), but the animals expressing the phospho-mimicking mutant displayed a mean seam cell number that is much closer to wt than S992A animals.…”

Vps54 regulatesDrosophilaneuromuscular junction development and controls postsynaptic density composition via a Rab7-dependent mechanism

“…Rab7 has also been shown to be required for exosome biogenesis and the release of exosomes, including those that containing miRNAs (Corrigan et al, 2014; Jae et al, 2015). Interestingly, the GARP component vps-52 regulates miRNA-mediated gene silencing by modulating levels of the miRNA pathway protein GW182 in C. elegans (Vasquez-Rifo et al, 2013). This raises the interesting possibility that the synergistic effects we observe in C380>scat shRNA, Rab7 (DN) double mutant NMJs may be due to defects in the production and secretion of exosomes containing signaling molecules or miRNAs from presynaptic terminals.…”