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A nuclear pore-anchored condensate enables germ granule organization and transgenerational epigenetic inheritance

Nature Structural & Molecular Biology volume 32, pages 1241–1254 (2025)Cite this article

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Abstract

Biomolecular condensates, such as stress and germ granules, often contain subcompartments. For instance, the Caenorhabditis elegans germ granule, which localizes near the outer nuclear membrane of germ cell nuclei, is composed of at least four ordered compartments, each housing distinct sets of proteins and RNAs. How these compartments form and why they are spatially ordered remains poorly understood. Here, we show that the conserved DEAD-box RNA helicase DDX-19 defines another compartment of the larger C. elegans germ granule, which we term the D compartment. The D compartment exhibits properties of a liquid condensate and forms between the outer nuclear pore filament and other compartments of the germ granule. Two nuclear pore proteins, NPP-14 and GLEL-1, are required for its formation, suggesting that the D compartment localizes adjacent to the outer nuclear membrane through interactions with the nuclear pore. The loss of DDX-19, NPP-14 or GLEL-1 leads to functional defects, including aberrant formation of the other four germ granule compartments, a loss of germline immortality and dysregulation of small RNA-based transgenerational epigenetic inheritance programs. Hence, we propose that a function of the D compartment is to anchor larger germ granules to nuclear pores, enabling germ granule compartmentalization and promoting transgenerational RNA surveillance.

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Fig. 1: Identification of a conserved RNA helicase DDX-19 that regulates RNAi inheritance and germline immortality.
Fig. 2: DDX-19 marks a liquid-like condensate termed the D compartment, which localizes between the P compartment and the nuclear pore.
Fig. 3: DDX-19 helicase activity promote D-compartment assembly in germ cells.
Fig. 4: D compartments are anchored by nucleoporins NPP-14 and GLEL-1.
Fig. 5: DDX-19, NPP-14 and GLEL-1 are dispensable for bulk mRNA export.
Fig. 6: DDX-19, NPP-14 and GLEL-1 regulate multiphase condensates homeostasis.
Fig. 7: DDX-19, NPP-14 and GLEL-1 regulate endogenous pUG RNA distribution and siRNA production.

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Data availability

The MS proteomics data were deposited to the ProteomeXchange Consortium through the PRIDE partner repository with dataset identifier PXD056752. pUG RNA-seq datasets and small RNA-seq datasets were deposited to the NCBI Sequence Read Archive under BioProject accession numbers PRJNA1171951 and PRJNA1171955. Source data are provided with this paper.

Code availability

Custom scripts are available from GitHub (https://github.com/WANLAB20192/Code_DDX-19_paper).

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Acknowledgements

We thank previous and present G.W. lab members for development and critical discussion of this work. We thank the company NanoInsights for providing the Multi-SIM microscope. We appreciate the support of J. L Xu (Core Facilities of Life Sciences, School of Life Sciences, Sun Yat‑sen University) for equipment support and technical assistance. This work was supported by the National Natural Science Foundation of China (32370729 and 32070798, to G.W.), the Shenzhen Medical Research Fund (B2302029, to G.W.) and the Guangdong Science and Technology Department (2023B1212060028). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Pu Lu, Boyuan Deng.

Authors and Affiliations

  1. Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Pu Lu, Boyuan Deng, Xinru Li, Xufang Niu, Yanhong Qiu, Yuntao Liang, Yonglin Liang, Guorun Tang, Zhongping Yuan, Guanzheng Luo & Gang Wan

  2. Innovation Center for Evolutionary Synthetic Biology, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    Pu Lu, Boyuan Deng, Xinru Li, Xufang Niu, Yanhong Qiu, Yuntao Liang, Yonglin Liang, Zhongping Yuan & Gang Wan

  3. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Scott Kennedy

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Contributions

P.L. and B.Y.D. conducted the majority of the experiments and analyses with assistance from X.F.N., Y.H.Q., Y.T.L., Y.L.L. and Z.P.Y. X.R.L. performed the bioinformatic analysis of small RNA-seq data. G.R.T. and G.Z.L. contributed to the development of the bioinformatic analysis strategy. S.K. contributed to the editing the manuscript. G.W. conceptualized and supervised the project, interpreted the results and wrote the paper.

Corresponding author

Correspondence to Gang Wan.

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Nature Structural & Molecular Biology thanks Carolyn Phillips, Ekaterina Voronina and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editors: Dimitris Typas and Sara Osman, in collaboration with the Nature Structural and Molecular Biology team.

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Extended data

Extended Data Fig. 1 DDX-19, GLEL-1, and NPP-14 are required for RNAi inheritance targeting two somatically expressed genes.

a-b, Animals with indicated genotype were subjected to lin-15b RNAi for two generations, representative images showing F1 adult animals (a), quantification of multi-vulva animals in F1 generation (b). The white arrow indicates extruded multi-vulva. Scale bar, 100 μm. n = 3 biological replicates (For each replicate, 50 animals were scored). These experiments were performed in an eri-1(mg366) background. Student’s t-test, two-tailed. Error bar, ±s.d. c-f, animals with indicated genotype were treated with control RNAi (L4440) or dpy-11 RNAi, the progeny was grown on OP50 plates. Representative images of F1 L4 larval stage animals were shown (c, d). Body length of F1 L4 larval stage animals was measured using ImageJ (e, f). Scale bar, 100 μm. n = 3 biological replicates (For each replicate, 10 animals were scored). One-way ANOVA. Error bar, ±s.d. g, Animals with indicated genotype were subjected to lin-15b RNAi for two generations, quantification of multi-vulva animals in F1 generation. These experiments were performed in an eri-1(mg366) background. n = 4 biological replicates. Student’s t-test, two-tailed. Error bar, ±s.d. h, Animals with indicated genotype were subjected to dpy-11 RNA inheritance assay. Percentage of animals with Dumpy phenotype in F1 L4 stage was scored. n = 4 biological replicates (for each replicate, 50 animlas were scored). Student’s t-test, two-tailed. Error bar, ±s.d. Source numerical data are available.

Source data

Extended Data Fig. 2 The TEI of RNAi targeting three endogenously expressed genes was not enhanced following the loss of DDX-19.

WT and ddx-19(-) animals were subjected to oma-1 RNAi (a), mex-6 RNAi(b), and puf-5 RNAi (c) treatment. Animals from P0, F1, F2, F4, F6, and F8 generations were collected and total RNAs were extracted. Quantitative reverse transcription and PCR (qRT-PCR) were employted to detect the mRNA levels of oma-1, mex-6, or puf-5 mRNAs in the indicated generations. n = 3 biological replicates. Student’s t-test, two-tailed. Error bar, ±s.d. Source numerical data are available.

Source data

Extended Data Fig. 3 Introduce fluorescence protein genes to endogenous locus generated functional fusion proteins.

a, WT, ddx-19(-) and ddx-19::gfp animals were grown on dpy-11 RNAi, F1 progeny were grown on OP50. The percentage of Dumpy animals in inheriting F1 was scored at L4 larval stage. n = 4 biological replicates. Error bar, ±s.d. Student’s t-test, two-tailed. b, WT, npp-14(-), npp-14::mCherry::ha and npp-14::gfp::3xflag animals were grown at 25 °C, brood size was counted at indicated generations. n = 3 biological replicates. Error bar, ±s.d. c, WT, glel-1(-) and ha::mCherry::glel-1 animals were subjected to dpy-11 RNAi treatment, percentage of Dpy animals were scored at F1 generation. n = 3 biological replicates. Error bar, ±s.d. d-e, WT, mut-2(-) and mut-2::mCherry animals (d) or WT, rsd-2(-) and rsd-2::gfp animals (e) were treated with pos-1 RNAi. The percentage of hatched animals was scored. n = 3 biological replicates. Error bar, ±s.d. Student’s t-test, two-tailed. Source numerical data are available.

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Extended Data Fig. 4 Genetic requirements of DDX-19::GFP marked D compartment.

a-b, Fluorescence imaging of animals expressing DDX-19::GFP. White arrows indicate DDX-19::GFP in somatic cells. Scale bar, 5 μm for images in (a), 10 μm for images in (b). n > 3 animals. c, Fluorescence imaging of animals expressing PGL-1::TagRFP in WT, glh-1(-), and deps-1(-) animals. Scale bar, 5 μm. n > 3 animals. d-e, Fluorescence imaging of animals expressing TagRFP::ZNFX-1 (d) or DDX-19::GFP (e) in WT and zsp-1(-) animals. Scale bar, 5 μm. n > 3 animals. f, Fluorescence imaging of animals expressing GFP::ZNFX-1 in WT and lotr-1(-) animals. Scale bar, 5 μm. n > 3 animals. g, Fluorescence imaging of animals expressing MUT-2::mCherry in WT and mut-16(-) animals. Scale bar, 5 μm. n > 3 animals.

Extended Data Fig. 5 The effect of genetic mutations on protein levels.

a, Schematic diagram of ddx-19 isoforms. IDR(+) indicates DDX-19 isoform 1 with complete IDR, IDR(-) indicates isoform 2-5 with truncated IDR or no IDR. b-c, Western blot to detect DDX-19::GFP isoforms in WT and DDX-19 helicase mutant young adult animals (b), quantification of DDX-19::GFP protein level normalized to Tubulin in (c). n = 3 biological replicates. Error bar, ±s.d. Student’s t-test, two-tailed. d-e, Western blot to detect DDX-19::GFP isoforms in WT and npp-14(-) young adult animals (d), quantification of DDX-19::GFP protein level normalized to Tubulin in (e). n = 3 biological replicates. Error bar, ±s.d. Student’s t-test, two-tailed. f-i, Western blot to detect DDX-19::GFP isoforms in WT young adult animals or embryos and young adult animals or embryos expressing NPP-14 with indicated residues mutated (f, h), quantification of DDX-19::GFP protein level normalized to Tubulin in (g, i). n = 3 biological replicates. Error bar, ±s.d. Student’s t-test, two-tailed. j-m, Western blot to detect DDX-19::GFP isoforms in WT, glel-1(-) and npp-26(-) young adult animals (j) and embryos (l), quantification of DDX-19::GFP protein level normalized to Tubulin in (k, m). n = 3 biological replicates. Error bar, ±s.d. Student’s t-test, two-tailed. n-q, Western blot to detect mCherry::GLEL-1 in WT, ddx-19(-) and npp-14(-) young adult animals (n) and embryos (p), quantification of mCherry::GLEL-1 protein level normalized to Tubulin in (o, q). n = 3 biological replicates. Error bar, ±s.d. Student’s t-test, two-tailed. Source numerical data and unprocessed blots are available.

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Extended Data Fig. 6 The localization interdependence of DDX-19, NPP-14 and GLEL-1.

a, Fluorescence imaging of embryos expressing DDX-19::GFP in npp-14 mutants background. Scale bar, 5 μm. n > 3 animals. b-c, Fluorescence imaging of WT(b) and npp-14(-) (c) animals expressing DDX-19::GFP and PGL-1::TagRFP. n > 3 animals. d, Fluorescence imaging of animals expressing NPP-14::mCherry. Scale bar, 10 μm. White arrows indicate NPP-14::mCherry in somatic cells. n > 3 animals. e, Fluorescence imaging of animals expressing mCherry::GLEL-1. Scale bar, 10 μm. White arrows indicate mCherry::GLEL-1 in somatic cells. n > 3 animals. f, A schematic diagram of the C. elegans tissues nuclear pore. g, Spinning disc confocal imaging of animals expressing indicated proteins. Scale bar, 1 μm. n > 3 animals. h, Fluorescence imaging of WT, ddx-19(-), npp-14(-), and glel-1(-) animals expressing NPP-21::GFP, NPP-1::GFP, or TagRFP::NPP-9. Scale bar, 5 μm. n > 3 animals. i, Fluorescence imaging of animals expressing NPP-14::mCherry in WT or ddx-19(-) background. Scale bar, 5 μm. n > 3 animals. j, Fluorescence imaging of animals expressing NPP-14::mCherry in WT or glel-1(-) background. Scale bar, 5 μm. n > 3 animals.

Extended Data Fig. 7 The effect of DDX-19, NPP-14, or GLEL-1 depletion on RNA export, PZMS condensates organization, and marker proteins level.

a-b, Poly(A)+ RNAs in animals expressing PGL-1::GFP in WT or ddx-19(-) background were detected with RNA FISH. Scale bar, 5 μm for images in (a) and 10 μm for images in (b). n > 3 animals. c-d, Fluorescence imaging of animals expressing SIMR-1::TagRFP in WT, ddx-19(-), npp-14(-), and glel-1(-) background (c). Image J analysis to measure the size of SIMR-1 marked SIMR foci in (d). The y axis is expressed in log2 scale. n = 3 animals (for each animal, all granules surrounding a dozen pachytene region nuclei was quantified). Student’s t-test, two-tailed. Error bar, ±s.d. e, Fluorescence imaging of PGL-1::TagRFP, GFP::ZNFX-1, MUT-16::GFP or SIMR-1::TagRFP in rachis of germline in animals with indicated genotype. The white arrows indicate granules in the rachis. Scale bar, 5 μm. n > 3 animals. f-m, Western blotting and ImageJ quantification of GFP::ZNFX-1 and PGL-1 (f-i), MUT-16::GFP (j-k) or SIMR-1::TagRFP (l-m) after loss of DDX-19, NPP-14 or GLEL-1. n = 3 biological replicates. Error bar, ±s.d. Student’s t-test, two-tailed. n, Fluorescence imaging of another PZMS marker proteins in WT, ddx-19(-), npp-14(-) or glel-1(-) background. Scale bar, 5μm. n > 3 animals. Source numerical data and unprocessed blots are available.

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Extended Data Fig. 8 Increased mixing of P and Z compartment components after loss of DDX-19, NPP-14, and GLEL-1 related to Fig. 6.

a-b, Fluorescence imaging of animals expressing GFP::ZNFX-1 and PGL-1::TagRFP in WT, ddx-19(-), npp-14(-), and glel-1(-) background (a). Image J analysis to measure the degree of colocalization between GFP::ZNFX-1 and PGL-1::TagRFP in WT and mutant animals (b). Scale bar, 1 μm for cells, 0.5 μm for inset. n = 27 (3 animals per biological replication, 3 nuclei per animal, 3 granules per nuclei). Error bar, ±s.d. Student’s t-test, two-tailed. c-d, Fluorescence imaging of animals expressing MUT-16::GFP and PGL-1::TagRFP (c) or MUT-16::GFP and TagRFP::ZNFX-1(d) in WT, ddx-19(-), npp-14(-) and glel-1(-) background. n > 3 animals. e, Fluorescence imaging of PGL-1::mCardinal, TagRFP::ZNFX-1, and MUT-16::GFP in WT, ddx-19(-), npp-14(-) and glel-1(-) animals. Scale bar, 1 μm for cells, 0.5 μm for inset. n > 3 animals. f, Quantification of the degree of colocalization in (e). n = 27 (3 animals, 3 nuclei per animal, 3 granules per nuclei). Error bar, ±s.d. Student’s t-test, two-tailed. g, ddx-19, npp-14 or glel-1 deletion were generated in animals expressing PGL-1::mCardinal, TagRFP::ZNFX-1 and MUT-16::GFP using CRISPR/Cas9. Fluorescence imaging was performed on animals expressing indicated proteins immediately at F2 generation when the heterozygous deletions became homozygous. n > 3 animals. h, Fluorescence imaging of animals expressing DDX-19::GFP and MUT-2::mCherry in WT, npp-14(-), and glel-1(-) background. n > 3 animals. i, Quantification of sphericity of DDX-19::GFP marked D compartment, PGL-1::mCardinal marked P compartment, and TagRFP::ZNFX-1 marked Z compartment in WT animals expressing DDX-19::GFP, PGL-1::mCardinal, and TagRFP::ZNFX-1. n = 27 (3 animals, 9 granule per germline). Student’s t-test, two tailed. Error bar, ±s.d. Source numerical data are available.

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Extended Data Fig. 9 pUG RNAs level, length and distribution are altered after loss of ddx-19, npp-14, or glel-1 related to Fig. 7.

a, pUG PCR to detect gfp pUG RNAs in indicated generations after animals were exposed gfp RNAi. n = 3 biological replicates. b, Strategy to detect pUG RNAs using next-generation sequencing. Total RNA was extracted, and reverse transcription was performed using an (AC)9 oligo plus adaptors to generate pUG cDNA. Three rounds of PCR were subsequently conducted. In the first round, PCR was performed using a gene-specific primer and an adaptor-specific primer. For the second round of PCR, the products from the first PCR were diluted 100-fold. PCR was then performed using library primer forward 1 containing the self-index and unique molecular identifier (UMI) and library primer reverse 1. After a second 100-fold dilution, the third PCR round was performed using library primer forward 2 and library primer reverse 2 containing Illumina p5 and p7 sequences, respectively. The final PCR products were pooled, resolved on an agarose gel, and purified. The purified DNA libraries were sequenced to generate paired-end 150 bp reads. UMI, Unique Molecular Identifier. Self-index, index used for distinguishing samples. c-d, An independent replicate to detect gfp pUG RNAs in indicated generations related to Fig. 7a. e, An independent replicate to detect gfp pUG RNA tail length in indicated generations related to Fig. 7b. f, Quantification of the volume of Z compartments with or without pUG RNAs near or within. n = 27 (3 animals per biological replication, 3 nuclei per animal, 3 granules per nuclei). Error bar, ±s.d. Student’s t-test, two-tailed. g, ddx-19, npp-14, or glel-1 deletion were generated in animals expressing GFP::ZNFX-1 using CRISPR/Cas9. RNA FISH was performed to detect endogenous pUG RNAs and poly(A)+ RNAs immediately at F2 generation when the heterozygous deletions became homozygous. n > 3 animals. Source numerical data and unprocessed gels are available.

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Extended Data Fig. 10 Features of DDX-19, NPP-14, and GLEL-1 Regulated Small RNAs and Working Model.

(a) Analysis of small RNA length and first nucleotide distribution in WT, ddx-19(-), npp-14(-), and glel-1(-) animals. (b) Biotype analysis of genes with significantly upregulated or downregulated targeting small RNAs in ddx-19(-), npp-14(-), and glel-1(-) animals compared with WT. (c) Representative factor analysis of genes with altered small RNAs in ddx-19(-), npp-14(-), and glel-1(-) animals against CSR-1, HRDE-1, and WAGO target lists. (d) piRNA target analysis of genes with altered small RNAs in ddx-19(-), npp-14(-), and glel-1(-) animals compared with WT. Welch’s t-test. (e) Comparison of genes with altered small RNAs in ddx-19(-), npp-14(-), and glel-1(-) animals with those in prg-1(-) or mut-16(-) mutants18. (f) Working Model. Biomolecular condensates are networks formed through protein-protein and protein-RNA interactions. Their organization depends on competing protein-protein interactions70. Homotypic interactions drive liquid-liquid phase separation, while non-competing heterotypic interactions result in co-phase separation, and competing heterotypic interactions lead to multiphase condensates71. In WT animals, the germline D compartment is anchored to the nuclear pore by NPP-14 and GLEL-1 at the cytoplasmic filament. This compartment is localized at the base of PZMS compartments, where NPP-14/GLEL-1 and P granules compete for DDX-19 interaction, facilitating D compartment formation. Loss of NPP-14 or GLEL-1 disrupts DDX-19 interactions, causing the D compartment to mix with P or Z compartments. This destabilizes the PZMS compartments, enlarging them and increasing local ZNFX-1 concentration. The elevated ZNFX-1 levels interact with more pUG RNAs, redistributing them from the M compartment to the mixed compartment and extending transgenerational epigenetic inheritance. This graph was created in BioRender. Deng, B. (2025) https://BioRender.com/w67i892.

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Lu, P., Deng, B., Li, X. et al. A nuclear pore-anchored condensate enables germ granule organization and transgenerational epigenetic inheritance. Nat Struct Mol Biol 32, 1241–1254 (2025). https://doi.org/10.1038/s41594-025-01515-7

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