RNA granules are protein/RNA condensates. How specific mRNAs are recruited to cytoplasmic RNA granules is not known. Here, we characterize the transcriptome and assembly of P granules, RNA granules in the C. elegans germ plasm. We find that P granules recruit mRNAs by condensation with the disordered protein MEG-3. MEG-3 traps mRNAs into non-dynamic condensates in vitro and binds to ~500 mRNAs in vivo in a sequence-independent manner that favors embryonic mRNAs with low ribosome coverage. Translational stress causes additional mRNAs to localize to P granules and translational activation correlates with P granule exit for two mRNAs coding for germ cell fate regulators. Localization to P granules is not required for translational repression but is required to enrich mRNAs in the germ lineage for robust germline development. Our observations reveal similarities between P granules and stress granules and identify intrinsically-disordered proteins as drivers of RNA condensation during P granule assembly.

Highlights d A mutant defective in germ granule assembly is deficient in RNAi d RNAi-deficiency correlates with small RNA silencing of genes required for RNAi d Silencing requires the piRNA Argonaute PRG-1 and the nuclear Argonaute HRDE-1 d Localization in germ granules protects transcripts from piRNA-initiated silencing

15P granules are perinuclear condensates in C. elegans germ cells proposed to serve as hubs for 16 self/non-self RNA discrimination by Argonautes. We report that a mutant (meg-3 meg-4) that does not 17 assemble P granules in primordial germ cells loses competence for RNA-interference over several 18 generations and accumulates silencing small RNAs against hundreds of endogenous genes, including the 19 RNA-interference genes rde-11 and sid-1. In wild-type, rde-11 and sid-1 transcripts are heavily targeted 20 by piRNAs, accumulate in P granules, but maintain expression. In the primordial germ cells of meg-3 21 meg-4 mutants, rde-11 and sid-1 transcripts disperse in the cytoplasm with the small RNA biogenesis 22 machinery, become hyper-targeted by secondary sRNAs, and are eventually silenced. Silencing requires 23 the PIWI-class Argonaute PRG-1 and the nuclear Argonaute HRDE-1 that maintains trans-generational 24 silencing of piRNA targets. These observations support a "safe harbor" model for P granules in 25 protecting germline transcripts from piRNA-initiated silencing. 26 27In the germ cells of animals, dense RNA-protein condensates accumulate on the cytoplasmic 29 face of the nuclear envelope. These condensates, collectively referred to as nuage, contain components 30 of the small RNA (sRNA) machinery that scan germline transcripts for foreign sequences. For example, in 31Drosophila, components of the piRNA machinery in nuage amplify small RNAs that target transcripts 32 from transposable elements for destruction (Huang et al., 2017). In C. elegans, the PIWI-class Argonaute 33 PRG-1 associates with ~15,000 piRNAs encoded in the genome that scan most, if not all, germline 34 1). Segregation of Argonautes and proteins required for 22G-RNA production into distinct nuage 60 compartments (P granules versus Z granules and mutator foci) could also play a role in sorting 22G-RNAs 61 or limiting their production (Wan et al., 2018). A direct test of these hypotheses, however, has been 62 difficult to obtain as complete loss of P granules causes sterility. 63We previously identified a mutant that affects P granule coalescence only during embryogenesis 64 (Wang et al., 2014). MEG-3 and MEG-4 are intrinsically-disordered proteins present in the germ plasm, a 65 specialized cytoplasm that is partitioned with the germ lineage during early embryonic cleavages (Wang 66 3 and Seydoux, 2013). MEG-3 and MEG-4 form gel-like scaffolds that recruit and stimulate the coalescence 67 of P granule proteins in germ plasm to ensure their partitioning to the embryonic germline and the 68 primordial germ cells Z2 and Z3 (Fig. 1;Putnam et al., 2019). In meg-3 meg-4 embryos, P granules do not 69 coalesce in germ plasm, causing granule components to be partitioned equally to all cells and turned 70 over ( Fig. 1; Wang et al., 2014). Despite lacking P granules during embryogenesis, meg-3 meg-4 71 assemble P granules de novo when the primordial germ cells resume divisions in the first larval stage to 72 generate the ~ 2000 germ cells that constitu...