Figure 7.
mRNA regulates the recruitment of EPG-2 to PGL granules. (A and B) Purified GFP::EPG-2 (1 µM) protein coats the surface of PGL-1/-3/SEPA-1 condensates (3 µM for each; A). Adding 30 ng/μl mRNA greatly decreases the level of GFP::EPG-2 coated on the surface of PGL-1/-3/SEPA-1 condensates (B). (C) In GFP-Trap assays, the level of endogenous PGL-3 coimmunoprecipitated by GFP::SEPA-1 is decreased, while the level of endogenous EPG-2 precipitated by GFP::SEPA-1 is increased, in embryonic extracts pretreated with RNase A compared with control embryonic extracts. The level of endogenous EPG-2 and PGL-3 precipitated by GFP::SEPA-1 was normalized by GFP::SEPA-1 and set to 1.0 under normal conditions. (D and E) In control embryos at 26°C (D), EPG-2 aggregates are partially colocalized with PGL granules labeled by anti-SEPA-1. The colocalization is increased in ruvb-1(RNAi) embryos (E). (F) Quantification of the colocalization ratio of EPG-2 aggregates and PGL granules in control, snr-4(RNAi), ruvb-1(RNAi), and prp-19(RNAi) embryos at 26°C. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed for each genotype). Two-tailed, unpaired t test results: *P < 0.05, ***P < 0.001. (G–I) Compared with sept-6(tm6608) mutant embryos at 26°C (G), the % of EPG-2 aggregates that are colocalized with PGL granules is decreased in dcr-1(bp132); sept-6(tm6608) mutant embryos (H). (I) shows quantification. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed for each genotype). Two-tailed, unpaired t test results: ***P < 0.001. Embryos at the ∼100-cell stage are shown in D, E, G, and H. (J and K) Compared to epg-2(bp287) mutant embryos at 26°C (J), the number of PGL granules is reduced by ruvb-1(RNAi) (K). Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in J and K. (L) Quantification of the number of PGL granules per embryo in the indicated genetic backgrounds. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed for each genotype). Two-tailed, unpaired t test results: **P < 0.01, ***P < 0.001. (M) Model for the role of RNA in regulating the fates of PGL granules under normal growth conditions (20°C) and mild heat stress conditions (26°C). At 20°C, mRNAs and translation proteins (e.g., IFE-1) are excluded from PGL granules, which are efficiently degraded by autophagy. At 26°C, mRNAs, RNA control factors, and translation proteins (e.g., IFE-1) are targeted to PGL granules, protecting them from degradation to facilitate stress adaptation. mRNAs promote LLPS of PGL granules, maintain their liquidity, and reduce the recruitment of EPG-2. Scale bars: 5 µm for A, B, D, E, G, H, J, and K; 2 µm for enlarged images in A, B, D, E, G, and H. Source data are available for this figure: SourceData F7.

mRNA regulates the recruitment of EPG-2 to PGL granules. (A and B) Purified GFP::EPG-2 (1 µM) protein coats the surface of PGL-1/-3/SEPA-1 condensates (3 µM for each; A). Adding 30 ng/μl mRNA greatly decreases the level of GFP::EPG-2 coated on the surface of PGL-1/-3/SEPA-1 condensates (B). (C) In GFP-Trap assays, the level of endogenous PGL-3 coimmunoprecipitated by GFP::SEPA-1 is decreased, while the level of endogenous EPG-2 precipitated by GFP::SEPA-1 is increased, in embryonic extracts pretreated with RNase A compared with control embryonic extracts. The level of endogenous EPG-2 and PGL-3 precipitated by GFP::SEPA-1 was normalized by GFP::SEPA-1 and set to 1.0 under normal conditions. (D and E) In control embryos at 26°C (D), EPG-2 aggregates are partially colocalized with PGL granules labeled by anti-SEPA-1. The colocalization is increased in ruvb-1(RNAi) embryos (E). (F) Quantification of the colocalization ratio of EPG-2 aggregates and PGL granules in control, snr-4(RNAi), ruvb-1(RNAi), and prp-19(RNAi) embryos at 26°C. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed for each genotype). Two-tailed, unpaired t test results: *P < 0.05, ***P < 0.001. (G–I) Compared with sept-6(tm6608) mutant embryos at 26°C (G), the % of EPG-2 aggregates that are colocalized with PGL granules is decreased in dcr-1(bp132); sept-6(tm6608) mutant embryos (H). (I) shows quantification. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed for each genotype). Two-tailed, unpaired t test results: ***P < 0.001. Embryos at the ∼100-cell stage are shown in D, E, G, and H. (J and K) Compared to epg-2(bp287) mutant embryos at 26°C (J), the number of PGL granules is reduced by ruvb-1(RNAi) (K). Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in J and K. (L) Quantification of the number of PGL granules per embryo in the indicated genetic backgrounds. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed for each genotype). Two-tailed, unpaired t test results: **P < 0.01, ***P < 0.001. (M) Model for the role of RNA in regulating the fates of PGL granules under normal growth conditions (20°C) and mild heat stress conditions (26°C). At 20°C, mRNAs and translation proteins (e.g., IFE-1) are excluded from PGL granules, which are efficiently degraded by autophagy. At 26°C, mRNAs, RNA control factors, and translation proteins (e.g., IFE-1) are targeted to PGL granules, protecting them from degradation to facilitate stress adaptation. mRNAs promote LLPS of PGL granules, maintain their liquidity, and reduce the recruitment of EPG-2. Scale bars: 5 µm for A, B, D, E, G, H, J, and K; 2 µm for enlarged images in A, B, D, E, G, and H. Source data are available for this figure: SourceData F7.

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