SIMR-1 and ENRI-2 colocalize at somatic granules in embryos.

A. Summary of IP-mass spectrometry interactions detected between NRDE-3, ENRI-2, ENRI-1, and SIMR-1 from previously published studies (Chen and Phillips, 2024, Lewis et al., 2021). The number of replicates from which the interaction was detected relative to the total number of replicates performed is indicated.

B. Live imaging of GFP::3xFLAG::NRDE-3 and SIMR-1::mCherry::2xHA; ENRI-2::2xTy1::GFP embryos at different stages (4-cell, 8-cell, 28-cell, 100-cell, 200-cell, and comma). Boxes identify the location of Z2 and Z3 primordial germ cells, showing that SIMR-1 is present in germ granules while ENRI-2 is not. At least five individual embryos were imaged for each genotype and stage. Scale bars, 5 μm.

C. Box plot of SIMR-1::mCherry::2xHA granule number quantification at different embryonic stages (4-cell, 8-cell, 28-cell, 100-cell, and 200-cell). At least ten individual embryos at each stage were used for quantification. Each dot represents an individual embryo, and all data points are shown. Bolded midline indicates median value, box indicates the first and third quartiles, and whiskers represent the most extreme data points within 1.5 times the interquartile range. Lines connect the mean granule number for each stage, illustrating the change in number of SIMR-1 granules across the developmental stages of the embryo. Two-tailed t-tests were performed to determine statistical significance and p-values were adjusted for multiple comparisons. See Materials and Methods for a detailed description of quantification methods.

Unloaded NRDE-3 localizes to cytoplasmic granules with SIMR-1.

A. Live imaging of GFP::3xFLAG::NRDE-3 embryos in eri-1, rde-3, and nrde-3(HK-AA) mutants at 8-cell, 100-cell, and comma stage embryos. At least five individual embryos were imaged for each genotype and stage. Arrows point to granule localization of NRDE-3 in the 100-cell stage. Asterisks highlights the localization of NRDE-3 to the nucleus of the Z2 and Z3 primordial germ cells. Scale bars, 5 μm.

B. Box plot of GFP::3xFLAG::NRDE-3 granule number quantification in different mutants.

C. Box plot of GFP::3xFLAG::NRDE-3(HK-AA) granule number quantification at different embryonic stages. Lines connect the mean granule number (red dots) for each stage, illustrating the change in change in number of NRDE-3 granules across embryonic development.

D. Live imaging of SIMR-1::mCherry::2xHA; GFP::3xFLAG::NRDE-3(HK-AA) at 100-cell stage. Arrows point to examples of colocalization between SIMR-1 and NRDE-3(HK-AA). At least ten individual embryos were imaged. Scale bars, 5 μm.

For box plots in B and C, at least twelve individual embryos in each mutant were used for quantification. Each dot represents an individual embryo, and all data points are shown. Bolded midline indicates median value, box indicates the first and third quartiles, and whiskers represent the most extreme data points within 1.5 times the interquartile range. Two-tailed t-tests were performed to determine statistical significance and p-values were adjusted for multiple comparisons. See Materials and Methods for a detailed description of quantification methods.

SIMR-1 recruits ENRI-2 and then NRDE-3 to cytoplasmic granules.

A. Live imaging of GFP::3xFLAG::NRDE-3(HK-AA) embryos in simr-1 and enri-2 mutants, and GFP::3xFLAG::NRDE-3 embryos in a simr-1; eri-1 double mutant at 8-cell, 100-cell, and comma stages. Asterisk marks the nuclear localization of NRDE-3 visible in a primordial germ cell. At least five individual embryos were imaged for each genotype and stage. Scale bars, 5 μm.

B. Live imaging of ENRI-2::2xTy1::GFP embryos in a simr-1 mutant and SIMR-1::GFP::3xFLAG embryos in an enri-2 mutant. At least five individual embryos were imaged for each genotype and stage. Arrows point to examples of cytoplasmic SIMR-1 granules still visible in the enri-2 mutant. Box surrounds a primordial germ cell displaying germ granule localization of SIMR-1. Scale bars, 5 μm.

CSR and WAGO pathway proteins localize to distinct cytoplasmic granules.

A. Live imaging of SIMR-1::mCherry::2xHA; RDE-3::GFP embryo at 100-cell stage, showing that RDE-3 colocalizes with SIMR-1. At least five individual embryos were imaged for each genotype and stage. Arrowheads point to examples of colocalization between SIMR-1 and RDE-3 at cytoplasmic granules. Scale bars, 5 μm.

B. Live imaging of SIMR-1::GFP::3xFLAG; HA::EGO-1::mCherry::RRF-1 at 100-cell stage embryo, showing that RRF-1 colocalizes with SIMR-1. At least five individual embryos were imaged for each genotype and stage. Arrowheads point to examples of colocalization between SIMR-1 and RRF-1 at cytoplasmic granules. Scale bars, 5 μm.

C. Live imaging of mCherry::EGO-1::GFP::RRF-1 in a simr-1 mutant, showing that RRF-1 no longer associates with cytoplasmic granules, while EGO-1 remains associated with granules in the simr-1 mutant. At least five individual embryos were imaged. Arrowheads point to examples of cytoplasmic EGO-1 granules in a simr-1 mutant. Scale bars, 5 μm.

D. Live imaging of SIMR-1::mCherry::2xHA embryos in a mut-16 mutant at 8-cell, 100-cell, and comma stages. At least five individual embryos were imaged. Asterisks indicate spindle localization of SIMR-1 in a mut-16 mutant. Box highlights germ granule localization of SIMR-1 in a comma-stage, mut-16 mutant embryo. Scale bars, 5 μm.

E. Live imaging of GFP::3xFLAG::CSR-1 embryos at different stages (4-cell, 28-cell, 100-cell, 200-cell, and comma), shows that CSR-1 localizes to cytoplasmic granules in early embryos and is restricted to germ granules in late embryos. At least three individual embryos were imaged for each stage. Dotted white line marks perimeter of the embryo. Box marks germ granule localization of CSR-1. Scale bars, 5 μM.

F. Box plot quantifying GFP::3xFLAG::CSR-1 granules at different embryonic stages. At least ten embryos at each stage were used for quantification. Each dot represents an individual embryo, and all data points are shown. Bolded midline indicates median value, box indicates the first and third quartiles, and whiskers represent the most extreme data points within 1.5 times the interquartile range. Lines connect the mean granule number (red dots) at each stage, illustrating the change in number of CSR-1 granules across embryonic development. Two-tailed t-tests were performed to determine statistical significance and p-values were adjusted for multiple comparisons. See Materials and Methods for a detailed description of quantification methods.

G. Live imaging of mCherry::EGO-1; GFP::3xFLAG::::CSR-1 embryo at 28-cell stage, showing CSR-1 colocalization with EGO-1. At least ten individual embryos were imaged. Arrowheads point to examples of CSR-1 and EGO-1 colocalization at cytoplasmic granules. Scale bars, 5 μm.

H. Live imaging of SIMR-1::mCherry::2xHA; GFP::3xFLAG::CSR-1 embryo at 28-cell stage, showing the absence of colocalization between SIMR-1 and CSR-1 with occasional adjacent localization. At least ten individual embryos were imaged. Arrowheads point to examples of SIMR-1 and CSR-1 granules that do not colocalize. Insets display examples of SIMR-1 and CSR-1 granules that are found adjacent to each other. Dotted white line marks perimeter of embryo. Scale bars, 5 μm.

I. Model of CSR and SIMR granules in the somatic cells of C. elegans embryos. The RdRP EGO-1, which synthesizes CSR-class 22G-RNAs, localizes to CSR granules, where CSR-1 loading may take place. The RdRP RRF-1, along with RDE-3, ENRI-2, and unloaded NRDE-3 localize to SIMR granules. SIMR-1 and ENRI-2 recruits unloaded NRDE-3 to granule where RRF-1 may synthesize ERGO-dependent 22G-RNAs for loading into NRDE-3. After loading, NRDE-3 translocates to the nucleus and silences genes co-transcriptionally.

NRDE-3 switches small RNA targets during development.

A. Diagram of IP-sRNA seq on NRDE-3 early embryos (<=100-cell stage) and late embryos (>=300-cell). GFP::FLAG::NRDE-3 was immunoprecipitated from embryo lysate and its associated small RNAs were isolated for sequencing.

B. Box plots depicting log2(fold change small RNA abundance) in NRDE-3 IP compared to input for at least two biological replicates.

C. Normalized NRDE-3-bound small RNA read distribution across a CSR-target gene (ztf-27) and an ERGO-target gene (Y37E11B.2) in early embryos, late embryos, and young adults. One representative replicate is shown.

D. Normalized NRDE-3 IP compared to input small RNA reads in early embryos, late embryos, and young adults. CSR-target and ERGO-target genes are indicated in blue and red, respectively. One representative replicate is shown. Insets are pie charts describing numbers of CSR targets, ERGO targets, and other targets that are significantly enriched in the NRDE-3 IP. The enriched targets were defined as small RNAs with at least 2-fold enrichment in IP compared to input, average RPM >10, and p-values ≤0.05.

E. Normalized NRDE-3 IP compared to input small RNA reads in eri-1 mutant early and late embryos. CSR-target and ERGO-target genes are indicated in blue and red, respectively. One representative replicate is shown.

F. Box plots depicting log2(fold change small RNA abundance) in mutants compared to wild-type in early embryos for two or three biological replicates.

G. Normalized NRDE-3 IP compared to input small RNA reads in simr-1 mutant and enri-2 mutant late embryos. CSR-target and ERGO-target genes are indicated in blue and red, respectively. One representative replicate is shown.

H. Box plots depicting log2(fold change of ERGO-class small RNA abundance) in NRDE-3 IP compared to input in wild-type and mutants in late embryos for two or three biological replicates.

For box plots in B,F,H, bolded midline indicates median value, box indicates the first and third quartiles, and whiskers represent the most extreme data points within 1.5 times the interquartile range, excluding outliers. Two-tailed t-tests were performed to determine statistical significance and p-values were adjusted for multiple comparisons.

NRDE-3 associates with CSR-class 22G-RNA in early embryos.

A. Live imaging of GFP::3xFLAG::NRDE-3 in one-day-adult germlines for wild-type, eri-1, rde-3, and nrde-3(HK-AA) mutants, showing that NRDE-3 localizes to the nuclei of oocytes in wild-type, eri-1 mutant, and rde-3 mutants, and to the cytoplasm in the nrde-3(HK-AA) mutant. At least five individual gonads were imaged for each genotype. Dotted white line traces the proximal portion of the C. elegans gonad and outlines the individual oocytes. Scale bars, 25 μm.

B. Live imaging of one-day-adult germlines and 8-cell embryos for degron::EGO-1; GFP::3xFLAG::NRDE-3 in a rde-3 mutant with ethanol (top) and 4mM auxin (bottom) treatment, showing that loss of both WAGO-class and CSR-class 22G-RNAs (rde-3 mutant and degron-mediated EGO-1 depletion) leads to cytoplasmic localization of NRDE-3 in both oocytes and early embryos. At least five individual gonads and embryos were imaged for each treatment condition. Dotted white line traces the proximal portion of the C. elegans gonad and outlines the individual oocytes. Scale bars, 25 μm in adults and 5 μm in embryos.

C. Venn diagrams indicate overlap of NRDE-3 IP enriched targets in early embryos (this work), CSR-1 IP enriched targets in young adults (Nguyen et al., 2021), and CSR-1 IP enriched targets in embryos (Quarato et al., 2021).

D. Density plot of small RNA enrichment on CSR targets in CSR-1 IP (dark blue), NRDE-3 IP (light blue) in embryos (top) and adults (bottom). Transcription start site (TSS) to transcription end site (TES) were plotted using normalized small RNA reads. All replicates are shown as individual lines.

E. Box plots depicting normalized log2(fold change of small RNA abundance in IP vs input) in a NRDE-3 IP in early embryos and CSR IP in embryos for two or three biological replicates. All genes list includes all genes expressed in IP or input. Early degraded mRNAs are maternal mRNAs that show at least twofold reduction in mRNA levels in early embryos (4 to 20 cell-stage) compared to 1-cell embryos (Quarato et al., 2021). Late degraded mRNAs are maternal mRNAs that show stable levels of mRNAs in early embryos and at least twofold reduction in late embryos (more than 20-cell stage) (Quarato et al., 2021). Zygotic mRNAs are mRNAs that are not detectable in 1-cell embryos but accumulate in early and late embryos (Quarato et al., 2021).

F. Box plot quantifying the number of embryos laid per adult csr-1::degron or csr::degron, gfp::nrde-3(HK-AA) animal on 4mM auxin plate. At least 65 individuals from each strain were scored. Each dot represents an individual animal, and all data points are shown.

G. Box plot depicting log2(fold change of H3K9me3 level in IP vs input) in wild-type (grey) and nrde-3 mutant (green) mixed-stage embryos, indicating that the H3K9me3 level of NRDE-3 targets in early embryos are not affected in nrde-3 mutant. Anti-H3K9me3 ChIP-seq data was obtained from Padeken et al. (2021).

For box plots in E,F,G, bolded midline indicates median value, box indicates the first and third quartiles, and whiskers represent the most extreme data points within 1.5 times the interquartile range, excluding outliers. Two-tailed t-tests were performed to determine statistical significance and p-values were adjusted for multiple comparisons.

Model of the function of cytoplasmic granules in C. elegans development.

Model of NRDE-3, SIMR-1, and CSR-1 function during C. elegans development. In early embryos, CSR-1 and EGO-1 localize to CSR granules and synthesize CSR 22G-RNAs to slice and clear maternal mRNAs. NRDE-3 binds CSR 22G-RNA in the nucleus and may transcriptionally silence germline-expressed genes. During mid-embryogenesis (e.g. around the 100-cell stage), unloaded NRDE-3, ENRI-2, RRF-1, and RDE-3 are recruited to SIMR granules in somatic cells by SIMR-1, where ERGO-dependent 22G-RNAs are produced and loaded to NRDE-3. In late embryos, NRDE-3 binds ERGO-dependent 22G-RNAs and silences ERGO-target genes in the nucleus. In adult C. elegans, somatic localized NRDE-3 associates with ERGO-dependent 22G-RNAs to transcriptionally silence ERGO-target genes, while germline localized NRDE-3 associates with CSR-class 22G-RNAs, possibly for deposition into early embryos.