C. elegans germ granules require both assembly and localized regulators for mRNA repression - PubMed (original) (raw)

C. elegans germ granules require both assembly and localized regulators for mRNA repression

Scott Takeo Aoki et al. Nat Commun. 2021.

Abstract

Cytoplasmic RNA-protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein-RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1. PGL-1 tethering represses an mRNA reporter in vivo.

a Left, C. elegans adult hermaphrodite possesses two gonadal arms with proliferating germ cells at one end (asterisk) and differentiating gametes at the other. Gonads make sperm (blue) first and then oocytes (pink). Right, P granules (magenta) reside at the nuclear periphery of all germ cells until late oogenesis. Modified from. b Linear diagram of C. elegans PGL-1. N-terminal dimerization domain (NtDD, yellow), central dimerization domain (CDD, orange), C-terminal region (C-region), and RGG repeats (blue). c Protein–mRNA tethering assay. The reporter mRNA encodes GFP (green)-histone H2B and harbors three boxB hairpins in its 3′UTR; a ubiquitous germline promoter drives expression (see Methods). λN22 peptide (light blue) is inserted into PGL-1 with a SNAP tag (magenta). Binding of PGL-1::SNAP::λN22 to boxB hairpins recruits reporter mRNA. Modified from. d–g GFP reporter expression in germ cells of live animals. (d, e) Brightfield image. (f, g) GFP fluorescence (green); auto fluorescence (red). n, number of animals scored for GFP expression. %, germlines with detectable GFP. Scale bar, 10 μm, in (d) applies to (d–g). Fisher’s exact test of PGL-1::SNAP vs. PGL-1::SNAP::λN22/+ (_p_-value < 0.0001). h–k Representative images in fixed gonads. (h, i) GFP fluorescence (green). (j, k) SNAP staining (magenta) and DNA (DAPI, cyan). n, number of germlines scored for GFP expression. Fisher’s exact test of PGL-1::SNAP vs. PGL-1::SNAP::λN22/+ (_p_-value < 0.0001). Scale bar, 10 μm, in (h) applies to images. Scale bar, 10 µm. Figure 1 and Fig. 5 results were performed in parallel, and thus results from (e, g, i, k) are the same as reported in Fig. 5b, d, f, h.

Fig. 2

Fig. 2. Crystal structure of PGL-1 NtDD.

a Crystal structure of C. japonica PGL-1 NtDD to 1.5 Å. See Supplementary Table 1 for crystal structure data and model statistics. NtDD has four copies per asymmetric unit (ASU). Copies in yellow, brown, cyan, and salmon. Arrows indicate two pairs of subunit interfaces in the ASU. Red arrows highlight the interface relying on conserved amino acids (see text). Red arrow: interface buried surface area average, 874.1 Å2. Black arrow: interface buried surface area average, 572.2 Å2. Area calculated with PISA. b Enlarged image of a single NtDD.

Fig. 3

Fig. 3. NtDD dimerization and its role in PGL-1 self-assembly.

a Structural model of the NtDD dimer. Subunits in yellow and brown. b, c Enlargement of dimer interface (red box in a). PGL-1 amino acids (b) K126 and K129, and (c) R123 interact with apposing subunit side chains. Residue labels in yellow or brown to indicate their representative subunits. d–f Size exclusion chromatography and multi-angle light scattering (SEC-MALS) of recombinant PGL-1 NtDD (d) wild-type (44,170 (±6.509%) Da), (e) K126E K129E (23,740 (±2.455%) Da), and (f) R123E (23,210 (±2.099%) Da) proteins. Differential refractive index (left _y_-axis) in arbitrary units (blue). Molecular weight (MW, right _y_-axis) in dalton (Da, red). Wild-type protein measured the approximate size of a dimer, while both mutant proteins measured approximately as monomers. BSA control protein analyzed by SEC-MALS in Supplementary Fig. 5g. g Diagram of C. elegans PGL-1 C-terminally tagged with GFP. N-terminal dimerization domain (NtDD, yellow), central dimerization domain (CDD, orange), C-terminal region (C-region), RGG repeats (blue), and GFP (green). h–k Representative images of (h) GFP-tagged PGL-1, (i) GFP alone, and GFP-tagged PGL-1, (j) K126E K129E, and (k) R123E mutants expressed in Chinese Hamster Ovary (CHO) cells. Cell cultures were imaged live, and GFP-positive cells counted for the presence or absence of granules. Images show the majority result (percentages noted above image). Fisher’s exact test versus GFP: PGL-1 GFP (_p_-value < 0.0001), PGL-1 K126E K129E (_p_-value = 0.3958), PGL-1 R123E (>0.9999). Hoechst (DNA) in blue. GFP in green. Scale bar, 10 µm.

Fig. 4

Fig. 4. NtDD dimerization is critical for fertility and P granule formation in nematodes.

a Sites of SNAP tag insertion and missense mutations in C. elegans PGL-1. N-terminal dimerization domain (NtDD, yellow), central dimerization domain (CDD, orange), C-terminal region (C-region), SNAP (magenta), and RGG repeats (blue). b Fertility of SNAP-tagged PGL-1 animals. Percentages were obtained after scoring individuals for production of larval progeny after 5 days at either 20 or 25 °C. Statistics reported in Supplementary Fig. 6a. c–p Extruded adult germlines, fixed, stained, and imaged in same region of meiotic pachytene (see Supplementary Fig. 6b). (c–f) Representative images of SNAP staining to visualize PGL-1 expression and granule formation. SNAP in magenta, DNA (DAPI) in cyan. All images are partial Z-stacks to maximize visualization of P granules. Images were taken from germlines containing embryos; similar images were obtained from germlines lacking embryos (Supplementary Fig. 6f, g). (c) PGL-1::SNAP localizes to granules around nuclei (n = 49). (d) Control, wild-type animal lacking SNAP tag shows virtually no background staining (n = 20). (e) PGL-1(K126E K129E)::SNAP is diffuse (n = 38). (f) PGL-1(R123E)::SNAP is diffuse (n = 24). (g–p) Representative images showing localization of three P granule components in germ cells expressing either (g–k) PGL-1::SNAP (n = 20) or (l–p) PGL-1(K126E K129E)::SNAP (n = 14). (g, l) DNA (DAPI, cyan); (h, m) SNAP (PGL-1::SNAP or mutant, magenta); (i, n) V5 (PGL-3, green); (j, o) MYC (GLH-1, red); (k, p) Merge. Scale bar, 10 µm for all images, except 2.5-fold enlargements of nuclei in boxes placed outside main images. n = biologically independent animals examined over 2 independent experiments.

Fig. 5

Fig. 5. PGL-1 assembly is required for tethered mRNA reporter repression.

a Protein–mRNA tethering assay and PGL-1 assembly. To test the necessity of granule formation for mRNA repression, NtDD assembly mutations were added to PGL-1::SNAP::λN22 and germlines observed for GFP reporter expression. N-terminal dimerization domain (NtDD, yellow), central dimerization domain (CDD, orange), SNAP (magenta), λN22 (light blue) and RGG repeats (blue), and GFP (green). Modified from. b–e GFP reporter expression in germ cells of live animals. (b, c) Brightfield image. (d, e) GFP fluorescence (green); auto fluorescence (red). n, number of animals scored for GFP expression. Scale bar, 10 μm, in (b) applies to (b–e) images. f–i Representative images of PGL-1 granule formation, seen by SNAP staining (magenta) and GFP fluorescence (green) in fixed gonads. DNA (DAPI) in cyan. n, number of germlines scored for GFP expression. %, germlines with detectable GFP. Fisher’s exact test of PGL-1::SNAP::λN22 vs. PGL-1 (K126E K129E)::SNAP::λN22 (_p_-value < 0.0001). Scale bar, 10 μm, in (f) applies to (f–i) images. Figure 1 and Fig. 5 results were performed in parallel, and thus results from (b, d, f, h) are the same as reported in Fig. 1e, g, i, k.

Fig. 6

Fig. 6. PGL-1 and WAGO-1 assemble independently in P granules.

a–d Representative images showing localization of PGL-1 and WAGO-1 in germ cells expressing (a) PGL-1::SNAP, WAGO-1::3xV5 (n = 33). (b) PGL-1(R123E)::SNAP, WAGO-1::3xV5 without WAGO-1 puncta (11 of 22 germlines). (c) PGL-1(R123E)::SNAP, WAGO-1::3xV5 with WAGO-1 puncta (11 of 22 germlines). (d) PGL-1::SNAP, WAGO-1(null)::3xV5 (n = 29). DNA (DAPI, cyan); SNAP (PGL-1::SNAP or mutant, magenta); V5 (WAGO-1::3xV5 wild-type or null, yellow). Scale bar, 10 µm for all images, except 2.5-fold enlargements placed outside main images. n = biologically independent animals examined over 2 independent experiments.

Fig. 7

Fig. 7. Argonaute WAGO-1 is required for PGL-1 tethered mRNA reporter repression.

a Protein–mRNA tethering assay and WAGO-1. To test the necessity of WAGO-1 for mRNA repression, PGL-1::SNAP::λN22 germlines were analyzed for GFP reporter expression in the presence or absence of WAGO-1. N-terminal dimerization domain (NtDD, yellow), central dimerization domain (CDD, orange), SNAP (magenta), λN22 (light blue) and RGG repeats (blue), and GFP (green). Modified from. b–e GFP reporter expression in germ cells of live animals with (b, d) wild-type wago-1 or (c, e) wago-1 null. (b, c) Brightfield image. (d, e) GFP fluorescence (green); auto fluorescence (red). n, number of animals scored for GFP expression. Scale bar, 10 μm, in (b) applies to (b–e) images. f–i Representative images of fixed germlines with (f, h) wild-type wago-1 or (g, i) wago-1 null. (f, g) GFP fluorescence (green). (h, i) DNA (DAPI, cyan) and PGL-1 granule formation seen by SNAP staining (magenta). n, number of germlines scored for GFP expression. Fisher’s exact test of wago-1 wild-type vs. null (_p_-value < 0.0001). Scale bar, 10 μm, in (f) applies to images (f–i).

Fig. 8

Fig. 8. Model of P granule assembly and mRNA repression.

a P granules assemble at the nuclear pore with assembly-competent PGL-1 protein. PGL-1 is shown as a dimer for simplicity but multivalent PGL-1s likely form an oligomeric protein–RNA network via its N-terminal dimerization domain (NtDD, yellow) and central dimerization domain (CDD, orange). WAGO-1 (blue) binds to RNA (purple), as expected for an Argonaute, and WAGO-1-associated RNAs are repressed, potentially by mRNA turnover (purple). b When PGL-1 NtDD cannot dimerize, PGL-1 fails to properly assemble into P granules at the nuclear periphery. WAGO-1 assembles independently into P granules. PGL-1-associated, non-granular mRNAs (green) are available for translation (ribosomes, black). c In the absence of cytoplasmic Argonaute WAGO-1, PGL-1 proteins assemble into P granules normally with its associated mRNA (purple), but loss of WAGO-1 perturbs repression of P granule-localized transcripts. PGL-1’s liquid droplet properties permit diffusion of some transcripts (green) into the cytoplasm for translation. See text for further discussion.

References

    1. Banani SF, Lee HO, Hyman AA, Rosen MK. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 2017;18:285–298. doi: 10.1038/nrm.2017.7. - DOI - PMC - PubMed
    1. Woodruff JB, Hyman AA, Boke E. Organization and function of non-dynamic biomolecular condensates. Trends Biochem. Sci. 2018;43:81–94. doi: 10.1016/j.tibs.2017.11.005. - DOI - PubMed
    1. Wu H, Fuxreiter M. The structure and dynamics of higher-order assemblies: amyloids, signalosomes, and granules. Cell. 2016;165:1055–1066. doi: 10.1016/j.cell.2016.05.004. - DOI - PMC - PubMed
    1. Hyman AA, Weber CA, Julicher F. Liquid-liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 2014;30:39–58. doi: 10.1146/annurev-cellbio-100913-013325. - DOI - PubMed
    1. Buchan JR. mRNP granules. Assembly, function, and connections with disease. RNA Biol. 2014;11:1019–1030. doi: 10.4161/15476286.2014.972208. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources