Figure S3.
mRNA metabolism factors modulate autophagic degradation and mRNA recruitment of PGL granules in embryos laid under heat stress conditions, related toFigs. 3, 4, and 5. (A–D) Compared with control(RNAi)-treated atg-3 mutant embryos (A), snr-4(RNAi) (B), prp-19(RNAi) (C), or ruvb-1(RNAi) (D) fails to reduce the accumulation of GFP::PGL-3 granules in atg-3(bp412) mutant embryos at 26°C. (E and F) Compared with control(RNAi)-treated bec-1(bp613) mutant embryos (E), snr-4(RNAi) fails to suppress the accumulation of SQST-1::GFP aggregates in bec-1(bp613) hypomorphic mutant embryos at 26°C (F). Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in A–F. (G) Quantification of the number of SQST-1::GFP aggregates per embryo in the indicated genetic background. Data are shown as mean ± SEM (n = 3; n refers to the number of embryos analyzed for each genotype). Two-tailed, unpaired t test results: n.s.: no significant difference. (H–K) Compared with control(RNAi)-treated atg-3 mutant embryos at 26°C (H), much fewer poly(A) mRNA puncta are formed in atg-3 embryos with simultaneous snr-4(RNAi) (I), prp-19(RNAi) (J), and npp-9(RNAi) (K). The nuclear signal of poly(A) mRNA is dramatically increased in prp-19(RNAi) and npp-9(RNAi) embryos. The germ precursor cells Z2 and Z3 are highlighted in dashed circles. Embryos at the ∼100–200 cell stage are shown in H–K. The exposure time for poly(A) mRNA puncta in atg-3 mutants shown here was shorter than that shown in Fig. 2 C and Fig. 4 A to avoid overexposure in atg-3; snr-4(RNAi), prp-19(RNAi); atg-3 and npp-9(RNAi); atg-3 embryos. (L and M) Quantification of the number of somatic poly(A) mRNA puncta (L) and % of PGL granules positive for poly(A) mRNA per focal plane in the indicated genotypes at 26°C (M). Data are shown as mean ± SEM (n = 5; n refers to five images from the corresponding five embryos analyzed for each genotype). Two-tailed, unpaired t test results: **P < 0.01, ***P < 0.001. (N) sfGFP::PAB-1 is diffusely localized in the cytoplasm of C. elegans embryos at 26°C. An embryo at the ∼200 cell stage is shown. (O and P) A large number of poly(A) mRNA puncta accumulate in dcr-1(bp132) mutant embryos at 26°C. The puncta are colocalized with P granules in germ precursor cells (highlighted in dashed circles) and PGL granules in somatic cells. Quantification of the fluorescence intensity of poly(A) puncta in somatic cells in WT and dcr-1(bp132) mutant embryos at 26°C is shown in P. Data are shown as mean ± SEM (n = 24 for WT and n = 35 for dcr-1; n refers to the number of poly(A) mRNA puncta analyzed for each genotype). Two-tailed, unpaired t test result: **P < 0.01. An embryo at the ∼100–200 cell stage is shown in O. (Q–S)dcr-1(bp132) mutant embryos show accumulation of PGL granules at 26°C, detected by anti-SEPA-1, as in WT embryos. S shows the quantification of the number of PGL granules in WT and dcr-1(bp132) mutants at 26°C. Data are shown as mean ± SEM (n = 3; n refers to the number of embryos analyzed for each genotype). Two-tailed, unpaired t test result: n.s.: no significant difference. Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in Q and R. Scale bars: 5 µm for A–F, H–K, N, O, Q, and R; 2 µm for enlarged images in H–K and O.

mRNA metabolism factors modulate autophagic degradation and mRNA recruitment of PGL granules in embryos laid under heat stress conditions, related to Figs. 3, 4, and 5. (A–D) Compared with control(RNAi)-treated atg-3 mutant embryos (A), snr-4(RNAi) (B), prp-19(RNAi) (C), or ruvb-1(RNAi) (D) fails to reduce the accumulation of GFP::PGL-3 granules in atg-3(bp412) mutant embryos at 26°C. (E and F) Compared with control(RNAi)-treated bec-1(bp613) mutant embryos (E), snr-4(RNAi) fails to suppress the accumulation of SQST-1::GFP aggregates in bec-1(bp613) hypomorphic mutant embryos at 26°C (F). Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in A–F. (G) Quantification of the number of SQST-1::GFP aggregates per embryo in the indicated genetic background. Data are shown as mean ± SEM (n = 3; n refers to the number of embryos analyzed for each genotype). Two-tailed, unpaired t test results: n.s.: no significant difference. (H–K) Compared with control(RNAi)-treated atg-3 mutant embryos at 26°C (H), much fewer poly(A) mRNA puncta are formed in atg-3 embryos with simultaneous snr-4(RNAi) (I), prp-19(RNAi) (J), and npp-9(RNAi) (K). The nuclear signal of poly(A) mRNA is dramatically increased in prp-19(RNAi) and npp-9(RNAi) embryos. The germ precursor cells Z2 and Z3 are highlighted in dashed circles. Embryos at the ∼100–200 cell stage are shown in H–K. The exposure time for poly(A) mRNA puncta in atg-3 mutants shown here was shorter than that shown in Fig. 2 C and Fig. 4 A to avoid overexposure in atg-3; snr-4(RNAi), prp-19(RNAi); atg-3 and npp-9(RNAi); atg-3 embryos. (L and M) Quantification of the number of somatic poly(A) mRNA puncta (L) and % of PGL granules positive for poly(A) mRNA per focal plane in the indicated genotypes at 26°C (M). Data are shown as mean ± SEM (n = 5; n refers to five images from the corresponding five embryos analyzed for each genotype). Two-tailed, unpaired t test results: **P < 0.01, ***P < 0.001. (N) sfGFP::PAB-1 is diffusely localized in the cytoplasm of C. elegans embryos at 26°C. An embryo at the ∼200 cell stage is shown. (O and P) A large number of poly(A) mRNA puncta accumulate in dcr-1(bp132) mutant embryos at 26°C. The puncta are colocalized with P granules in germ precursor cells (highlighted in dashed circles) and PGL granules in somatic cells. Quantification of the fluorescence intensity of poly(A) puncta in somatic cells in WT and dcr-1(bp132) mutant embryos at 26°C is shown in P. Data are shown as mean ± SEM (n = 24 for WT and n = 35 for dcr-1; n refers to the number of poly(A) mRNA puncta analyzed for each genotype). Two-tailed, unpaired t test result: **P < 0.01. An embryo at the ∼100–200 cell stage is shown in O. (Q–S)dcr-1(bp132) mutant embryos show accumulation of PGL granules at 26°C, detected by anti-SEPA-1, as in WT embryos. S shows the quantification of the number of PGL granules in WT and dcr-1(bp132) mutants at 26°C. Data are shown as mean ± SEM (n = 3; n refers to the number of embryos analyzed for each genotype). Two-tailed, unpaired t test result: n.s.: no significant difference. Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in Q and R. Scale bars: 5 µm for A–F, H–K, N, O, Q, and R; 2 µm for enlarged images in H–K and O.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal