Germ Granules Coordinate RNA-based Epigenetic Inheritance Pathways

Abstract

Germ granules are biomolecular condensates that promote germ cell totipotency in most, if not all, animals. In C. elegans, MEG-3 and MEG-4 are two intrinsically disordered proteins that are redundantly required for the phase separations that drive germ granule assembly in germline blastomeres. Here, we show that animals lacking MEG-3/4 exhibit defects in dsRNA-mediated gene silencing (RNAi) that are due, at least in part, to defects in systemic RNAi. Interestingly, these RNAi defects are transgenerationally disconnected from meg-3/4 genotype: RNAi defects do not arise until 5-9 generations after animals become mutant for meg-3/4, and RNAi defects persist for 9-11 generations after meg-3/4 genotype is restored to wild type. Similar non-Mendelian patterns of inheritance are associated with other mutations that disrupt germ granule formation, indicating that germ granule disruption is the likely cause of genotype/phenotype disconnects. Loss of germ granules is associated with the production of aberrant populations of endogenous siRNAs, which, remarkably, are propagated for ≅10 generations in wild-type descendants of animals that lacked germ granules. sid-1, which encodes a factor required for systemic RNAi in C. elegans, is inappropriately and heritably silenced by aberrantly expressed sid-1 endogenous siRNAs, suggesting that transgenerational silencing of sid-1 likely underlies the heritable defect in RNAi. We conclude that one function of germ granules is to organize RNA-based epigenetic inheritance pathways and that failure to assemble germ granules has consequences that persist across many generations.3

ZSP‐1 is a Z granule surface protein required for Z granule fluidity and germline immortality in Caenorhabditis elegans

“…It is also possible that the role of Z granules in germline RNAi is indirect, as previous studies have shown that disrupting germ granules can indirectly affect RNAi responsiveness in C. elegans (Dodson & Kennedy, 2019; Ouyang et al , 2019; Lev et al , 2019). For instance, the intrinsically disordered proteins MEG‐3 and MEG‐4 (MEG‐3/4) promote P granule assembly and are, like ZSP‐1, required for germline RNAi (Dodson & Kennedy, 2019; Ouyang et al , 2019). The germline RNAi defect associated with meg‐3/4 is indirect in that RNAi defects are due to HRDE‐1‐dependent aberrant silencing of rde‐11 / sid‐1 and not because P granule formation is itself necessary for germline RNAi (Dodson & Kennedy, 2019; Ouyang et al , 2019).…”

“…For this reason, and because P granules localize directly adjacent to nuclear pores, it has been suggested that P granules are major sites of small RNA-directed mRNA surveillance in C. elegans germ cells (Wang & Reinke, 2008;Batista et al, 2008;Claycomb et al, 2009;Gu et al, 2009;Beshore et al, 2011). Consistent with this idea, mutations that disrupt P granule formation cause misexpression of somatic and sperm mRNAs within oogenic germlines and cause small regulatory RNA-based gene regulation to become disorganized and defective (Spike et al, 2008;Campbell & Updike, 2015;Spracklin et al, 2017;Knutson et al, 2017;Dodson & Kennedy, 2019;Ouyang et al, 2019). Other compartments of the perinuclear nuage are also thought to function in distinct, yet related aspects of small RNA-based gene regulation.…”

DEPS-1 is required for piRNA-dependent silencing and PIWI condensate organisation in Caenorhabditis elegans

“…However, it is only recently that a role for the P granule in ensuring appropriate small RNA-mediated silencing has been shown. Using mutant animals that lack P granules in the C. elegans primordial germ cells, these studies elegantly show that P granules are required for transgenerational inheritance of RNAi 44 , 50 , 51 . The direct interaction between PRG-1 and DEPS-1 provides an example for how small RNAs and P granules can be biochemically coupled.…”

“…6c). deps-1 also affects wago targets 44 (300 out of 425 wago targets show > twofold reduction in 22Gs level in deps-1 mutant; Hypergeometric Test: p < 10 −139 ; Fig. 4d and Supplementary Fig.…”

The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm

“…Recent studies in C. elegans have shown that piRNA/ PRG1-dependent mRNA accumulation in germ granules prevent their silencing, strengthening the function of piRNAs in germ granules for mRNA storage and surveillance. 56,57 In Drosophila, Aub mediates the link between piRNAs and mRNA regulation in germ granules since Aub localization to germ granules depends on its loading with piRNAs 12 and Aub/piRNAs play a general role in the localization and stabilization of germ cell mRNAs in germ granules. 13,19 How do germ granules accommodate translational control has remained more elusive.…”