Caenorhabditis elegans germ granules are present in distinct configurations that differentially associate with RNAi-targeted RNAs

Abstract

RNA silencing pathways are complex, highly conserved, and perform widespread, critical regulatory roles. In C. elegans germlines, RNA surveillance occurs through a series of perinuclear germ granule compartments-P granules, Z granules, SIMR foci, and Mutator foci-multiple of which form via phase separation and exhibit liquid-like properties. The functions of individual proteins within germ granules are well-studied, but the spatial organization, physical interaction, and coordination of biomolecule exchange between compartments within germ granule "nuage" is less understood. Here we find that key proteins are sufficient for compartment separation, and that the boundary between compartments can be reestablished after perturbation. Using super-resolution microscopy, we discover a toroidal P granule morphology which encircles the other germ granule compartments in a consistent exterior-to-interior spatial organization. Combined with findings that nuclear pores primarily interact with P granules, this nuage compartment organization has broad implications for the trajectory of an RNA as it exits the nucleus and enters small RNA pathway compartments. Furthermore, we quantify the stoichiometric relationships between germ granule compartments and RNA to reveal discrete populations of nuage that differentially associate with RNAi-targeted transcripts, possibly suggesting functional differences between nuage configurations. Together, our work creates a more spatially and compositionally accurate model of C. elegans nuage which informs the conceptualization of RNA silencing through different germ granule compartments.

Figure 1.. Mutator foci and P granule separation is independent of nuclear association and can be reestablished after perturbation.

(A) Widefield immunofluorescence of mut-16::mCherry; pgl-1::gfp germlines shows that endogenous Mutator foci (MUT-16, magenta), and P granules (PGL-1, green), are adjacent yet distinct compartments. (B) Ectopically expressed MUT-16::mCherry (magenta) and PGL-1::GFP (green) driven by the myo-3 muscle-specific promoter create condensates that maintain separation in the muscle environment. (C) Top: Live images of the transition zone of mut-16::mCherry; pgl-1::gfp gonads dissected in M9 buffer (Ci) or varying concentrations of 1,6-hexanediol (Cii-vi). Middle: Mutator foci (MUT-16, red) are dispersed in all concentrations of 1,6-hexanediol except 0.625%. Numbers indicate how many gonads displayed Mutator foci out of total assessed gonads. Bottom: P granules (PGL-1, green) are disrupted in only 10% and 5% 1,6-hexanediol, indicating different sensitivities to perturbation of weak hydrophobic interactions. Numbers indicate how many gonads displayed P granules out of total assessed gonads. (D) mut-16::mCherry; pgl-1::gfp animals were subjected to heat stress at 34 °C for 2 hours and allowed to recover at room temperature (~21 °C) for 2 hours. Top: Representative live images of the pachytene region were collected before heat stress (Di, no h.s.), immediately after heat stress (Dii, 0 min recovery), and for every 30 minutes during recovery (Diii-Dvi). Middle: Mutator foci (MUT-16, red) weakly colocalizes with P granules immediately after heat shock. At 30- and 60-minutes room temperature recovery, Mutator foci loses colocalization with P granules but remains dispersed in the cytoplasm. By 90- and 120- minutes room temperature recovery, MUT-16 reappears as separate, punctate foci adjacent to P granules, indicating the interaction is able to be re-established after perturbation. Numbers indicate gonads displaying punctate Mutator foci out of total assessed gonads. For Dii, colocalization with PGL-1 was seen in 3/3 gonads. Bottom: P granules do not completely disperse after 2 hours 34 °C heat stress. Numbers indicate how many gonads displayed P granules out of total assessed gonads. Scale bars, 5 µm.

Figure 2.. P granules form unique pocket morphologies in the mid and late pachytene.

3D-Structured Illumination Microscopy images of immunostained mut-16::mCherry; pgl-1::gfp germlines display P granules (PGL-1, green) and Mutator foci (MUT-16, magenta). (A) P granule and Mutator focus are adjacent to one another in the transition zone as previously described. (B) Some P granules appear to form an arc or circular morphology (arrows) around Mutator foci in the mid pachytene. (C) A toroidal, “donut-like” P granule morphology is readily apparent in the late pachytene (insets, arrow). Insets highlight the unique morphology, termed “P granule pocket”. Each P granule pocket appears to surround a Mutator focus, yet a gap is maintained between the foci. Images for (A) and (B) are comprised of 10 maximum projection Z-stacks (0.125 µm Z-step). Image (C) is comprised of 55 maximum-projection Z stacks (0.125 µm Z-step). Scale bars, 5 µm.

Figure 3.. Nuage compartments exhibit a hierarchical stoichiometry.

(A) Violin plot of fluorescently tagged germ granules surrounding nuclei in the late pachytene, with each dot corresponding to one nucleus (n = 30). Asterisks indicate average foci per nucleus. ** indicates p-value ≤ 0.01, **** indicates p-value ≤ 0.0001. Significance was determined with a two-tailed equal variance Student’s t-test. (B) Manual adjacency quantification from either mut-16::gfp; rfp::znfx-1; pgl-1::bfp to determine P granule populations (left) or mut-16::gfp simr-1::mCherry; pgl-1::bfp to determine SIMR foci populations (right). Overlapping pie charts reveal distinct populations of granule association. (C) Summary of combined granule stoichiometry indicating the percent that any one compartment (y-axis) is adjacent to a second compartment (x-axis). Of note, Mutator foci are always associated with all nuage compartments (PZM, n = 183 and PSM, n = 117), suggesting a hierarchical assembly of compartments. (D) Representative widefield image of a fixed mut-16::gfp; rfp::znfx-1; pgl-1::bfp late pachytene nucleus displaying the different P granule populations: P granule only (P, asterisk), P granule associated with Z granule (PZ, arrowhead), P granule associated with both Z granule and Mutator focus (PZM, arrow). Scale bar, 1 µm.

Figure 4.. P granule pockets exhibit an exterior-to-interior organization.

(A) Structured illumination of immunostained germlines with endogenously tagged SIMR-1 to detect SIMR foci (cyan), and ZNFX-1 to detect Z granules (magenta). P granules (yellow) are visualized with anti-PGL-1. Inset: a Z granule occupies the entire interior of the P granule pocket and a SIMR focus is innermost still. (B) Confocal image of fixed pachytene nuclei from a mut-16::gfp; tagRFP::znfx-1; pgl-1::bfp germline. Inset: a Z granule (magenta) encompasses a Mutator focus (cyan). (C) Structured illumination of immunostained germlines with endogenously tagged ZNFX-1 (magenta) and MUT-16 (green). Nuclear pore complexes (cyan) are visualized with anti-Nup 107 (mAb414). Inset: a Z granule occupies the interior of the nuclear pore pocket and a Mutator focus localizes to the center. (D) Confocal image of the late pachytene region of a fixed gfp::znfx-1, tagRFP::npp-9; pgl-1::bfp germline demonstrates nuclear pore interaction with germ granules. Scale bars, 1 µm.

Figure 5.. Silenced RNA associates preferentially with specific germ granule populations.

(A-B) Confocal image of three oocytes in the diakinesis region of a mut-16::gfp; pgl-1::bfp germline following 6 hours on control (L4440) RNAi (A) or mex-6 RNAi (B). Proximal oocytes are oriented to the right. (B) smFISH for both oma-1 (control) and mex-6 RNA shows that mex-6 RNA, and not oma-1 RNA, associates with germ granules after mex-6 RNAi. Scale bars, 5 µm. (C-D) Insets from (A) and (B) showing mex-6 RNA association with individual germ granules. For mex-6 RNAi-treated animals, the mex-6 RNA signal is located between the signals for PGL-1 and MUT-16. (E) Quantification of granules associated with RNA from mut-16::gfp; pgl-1::bfp following 6 hours on mex-6 RNAi to determine frequency of P and PM interactions with mex-6 RNA.

Figure 6.. Working model of P granule pocket organization and nuage assembly.

Top: Model of a P granule exhibiting toroidal “pocket” morphology at the periphery of a C. elegans germ cell nucleus (gray). Nuclear pores (dark gray) interact primarily with the P granule pocket (teal), enabling P granules to capture nascent RNA (black). The P granule pocket encircles a Z granule (red) which balances secondary siRNA synthesis across transcripts and is required for siRNA inheritance. The Z granule further encompasses a SIMR focus (purple), which acts as an intermediate between primary and secondary siRNA pathways, and a Mutator focus, which is required for secondary siRNA synthesis (orange). Bottom: Assembly hierarchy of P granule populations proceeding from P to PZSM (left to right). P granules associated with Mutator foci are associated with all known nuage compartments.