[
International Worm Meeting,
2021]
Argonaute proteins catalyze RNA-guided search to direct RNA interference (RNAi) and micro-RNA based gene silencing. These natural programmable search mechanisms have fundamental roles in gene regulation and development. In addition, a new class of precision medicines function by artificially programming Argonautes with synthetic guide RNAs (siRNAs) designed to target disease-related gene expression. Despite a great deal of knowledge and research on Argonaute mediated silencing, surprisingly little is known about how Argonaute proteins normally acquire and load their RNA guides. Understanding how Argonautes are properly loaded could provide novel insight into aberrant loading events in human diseases. Transgenerational silencing in the C. elegans germline depends on a constant cycle of de-novo Argonaute synthesis and loading necessary to generate robust supplies of Argonaute guide complexes that transmit silencing via the sperm and egg to offspring. Our previous investigation of this silencing process has identified protein candidates for factors that stabilize and promote the loading of unloaded Argonautes. One of these is GLH-1, where perturbation of the ATP binding domain increases affinity to unloaded WAGO-1. Here we design and validate unloadable mutations of WAGO-1 to further probe the role of the GLH-1 ATP binding cycle in loading WAGO-1. We report that unloadable mutations of WAGO-1 are stably expressed, with no signs of innate instability despite previous reports. We also find that inhibition of WAGO-1 loading and perturbation of GLH-1 ATP binding cycle work synergically to reveal a soluble, cytoplasmic complex of WAGO-1 and GLH-1. Future work will aim to leverage the unloadable mutations of WAGO-1 and other germline Argonautes to identify loading co-factors.
[
Biochem J,
2005]
Manganese is an essential, but potentially toxic, trace metal in biological systems. Overexposure to manganese is known to cause neurological deficits in humans, but the pathways that lead to manganese toxicity are largely unknown. We have employed the bakers'' yeast Saccharomyces cerevisiae as a model system to identify genes that contribute to manganese-related damage. In a genetic screen for yeast manganese-resistance mutants, we identified S. cerevisiae MAM3 as a gene which, when deleted, would increase cellular tolerance to toxic levels of manganese and also increased the cell''s resistance towards cobalt and zinc. By sequence analysis, Mam3p shares strong similarity with the mammalian ACDP (ancient conserved domain protein) family of polypeptides. Mutations in human ACDP1 have been associated with urofacial (Ochoa) syndrome. However, the functions of eukaryotic ACDPs remain unknown. We show here that S. cerevisiae MAM3 encodes an integral membrane protein of the yeast vacuole whose expression levels directly correlate with the degree of manganese toxicity. Surprisingly, Mam3p contributes to manganese toxicity without any obvious changes in vacuolar accumulation of metals. Furthermore, through genetic epistasis studies, we demonstrate that MAM3 operates independently of the well-established manganese-trafficking pathways in yeast, involving the manganese transporters Pmr1p, Smf2p and Pho84p. This is the first report of a eukaryotic ACDP family protein involved in metal homoeostasis.