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RNA recruitment switches the fate of protein condensates from autophagic degradation to accumulation - PubMed

  • ️Sun Jan 01 2023

RNA recruitment switches the fate of protein condensates from autophagic degradation to accumulation

Hui Zheng et al. J Cell Biol. 2023.

Abstract

Protein condensates can evade autophagic degradation under stress or pathological conditions. However, the underlying mechanisms are unclear. Here, we demonstrate that RNAs switch the fate of condensates in Caenorhabditis elegans. PGL granules undergo autophagic degradation in embryos laid under normal conditions and accumulate in embryos laid under heat stress conditions to confer stress adaptation. In heat-stressed embryos, mRNAs and RNA control factors partition into PGL granules. Depleting proteins involved in mRNA biogenesis and stability suppresses PGL granule accumulation and triggers their autophagic degradation, while loss of activity of proteins involved in RNA turnover facilitates accumulation. RNAs facilitate LLPS of PGL granules, enhance their liquidity, and also inhibit recruitment of the gelation-promoting scaffold protein EPG-2 to PGL granules. Thus, RNAs are important for controlling the susceptibility of phase-separated protein condensates to autophagic degradation. Our work provides insights into the accumulation of ribonucleoprotein aggregates associated with the pathogenesis of various diseases.

© 2023 Zheng et al.

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Conflict of interest statement

Disclosures: The authors declare no competing interests exist.

Figures

Figure S1.
Figure S1.

PGL granules formed under heat stress conditions contain components of the translation machinery, related to Fig. 1. (A) Proteomics data from three independent replicates were analyzed by volcano plot. Blue dots indicate proteins significantly enriched in the GFP::PGL-3 IP-MS compared to GFP control (log2 fold change ≥1 and two-tailed, unpaired t test results P < 0.05). The top 10 enriched proteins are highlighted. Gray dots indicate proteins with no significant enrichment. (B) Brief description of the top 10 enriched proteins identified in the GFP::PGL-3 IP-MS compared the GFP control. GLH-1 and GLH-2 are the known components of germline P granules. (C) The 574 proteins that are significantly enriched in the GFP::PGL-3 IP-MS compared with GFP control were analyzed by DAVID. The top GO enrichment terms for molecular function are listed according to the number of proteins with P < 0.05 (automatically generated by DAVID). Black bars indicate proteins involved in RNA-related processes. (D and E) In atg-3(bp412) mutant embryos, IFE-1::GFP forms a large number of granules in somatic cells. Compared with embryos grown at 20°C (D), the number and intensity of IFE-1::GFP granules are increased, and the level of diffuse IFE-1::GFP is decreased, in embryos grown at 26°C (E). (F and G) The number and intensity of IFE-1::GFP granules are reduced in sepa-1(bp1726); atg-3(bp412) double mutant embryos at both 20°C (F) and 26°C (G). Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in D–G. (H) Quantification of the number of IFE-1::GFP granules in somatic cells. 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: *P < 0.05. (I and J) Somatic IFE-1::GFP granules are partially colocalized with PGL granules labeled by TagRFP::SEPA-1 in atg-3(bp412) mutant embryos at 20°C (I) and 26°C (J). Some of the IFE-1::GFP granules are negative for SEPA-1. Comma-stage embryos are shown in I and J. (K and L) 3 µM purified TagRFP::IFE-1 protein fails to undergo LLPS during the observation time of 5 min. The reaction contained 150 mM NaCl. K is the DIC image of L. (M and N) TagRFP::IFE-1 is not evidently partitioned into droplets formed by 3 µM PGL-3/SEPA-1 proteins in an in vitro LLPS assay. M is the DIC image of N. Scale bars: 5 µm for D–G, I, and J; 2 µm for enlarged images in I and J; 20 µm for K–N.

Figure 1.
Figure 1.

PGL granules formed under heat-stress conditions contain proteins involved in translation. (A and B) IFE-1::GFP forms a few granules in the germ precursor cells (Z2 and Z3, highlighted in dashed circles), and is diffusely localized in the cytoplasm of somatic cells in wild-type (WT) embryos at 20°C. Embryos at the ∼100-cell stage and the comma stage are shown in A and B, respectively. (C and D) Temporal accumulation of IFE-1::GFP granules in somatic cells in WT embryos laid at 26°C. IFE-1::GFP forms a few tiny granules at the ∼100-cell stage (C), and forms a large number of granules at the comma stage (D). (E and F) IFE-1::GFP granules are absent from somatic cells in sepa-1(bp1726) (E) and pgl-1(RNAi) (F) embryos at the comma stage at 26°C. In pgl-1(RNAi) embryos, the number of IFE-1::GFP granules is also reduced in two-germ precursor cells (highlighted in dashed circles). Maximum-intensity projections of Z-stack confocal images are shown in A–F. (G) Quantification of the number of IFE-1::GFP granules in somatic cells per embryo at the ∼100-cell and comma stage at 20 and 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 results: ***P < 0.001. (H) IFE-1::GFP granules are colocalized with PGL granules, labeled by TagRFP::SEPA-1, in WT embryos laid at 26°C. PGL granules contain oocyte-derived PGL-1/-3 and the zygotically synthesized SEPA-1. PGL-1/-3 are present in both P granules and PGL granules, while SEPA-1 is present only in PGL granules. (I) Quantification of the percentage (%) of PGL granules positive for IFE-1::GFP in WT and atg-3 mutant embryos. Data are shown as mean ± SEM (n = 3; n refers to the number of embryos analyzed for each genotype or condition). Two-tailed, unpaired t-test results: **P < 0.01, ***P < 0.001. (J and K) Purified TagRFP::IFE-1 protein is partitioned into PGL-1/-3/SEPA-1 condensates (3 µM for each protein) in in vitro LLPS assays. J is the DIC image of K. His-tagged PGL-1 and PGL-3, His- and TagRFP-tagged IFE-1 isoform b, and His- and MBP-tagged SEPA-1 were used for LLPS in this study unless otherwise noted. Scale bars: 5 µm for A–F and H; 2 µm for enlarged images in H; 20 µm for J and K.

Figure 2.
Figure 2.

PGL granules act as sites for mRNA metabolism that are distinct from stress granules. (A) Distinct poly(A) mRNA puncta are absent from somatic cells of WT embryos at 20°C, but are detected in germ precursor cells (highlighted in dashed circles) where they colocalize with P granules. PGL proteins are quickly degraded by autophagy in WT embryos under normal growth conditions. Thus, PGL granules are largely absent in embryos at late developmental stages. A few small granules are detected at early embryonic stages. One focal plane in a ∼100-cell-stage embryo is shown in A. (B) A large number of puncta containing poly(A) mRNAs accumulate in WT embryos at 26°C. These puncta are colocalized with P granules in germ precursor cells (Z2 and Z3, highlighted in dashed circles) and PGL granules in somatic cells labeled by PGL-1::TagRFP. (C) Poly(A) mRNA puncta are largely absent from PGL granules in somatic cells in atg-3 mutant embryos at 20°C but are detected in germ precursor cells (highlighted in dashed circles). (D) In atg-3 mutant embryos at 26°C, multiple poly(A) mRNA puncta accumulate, which are colocalized with P granules in germ precursor cells (highlighted in dashed circles) and PGL granules in somatic cells. One focal plane in a ∼100–200 cell-stage embryo is shown in B–D. The poly(A) mRNA puncta in atg-3 mutants at 20°C are much weaker in intensity. (E and F) Quantification of the percentage (%) of PGL granules positive for poly(A) mRNA (E), and the number of poly(A) mRNA puncta (F) in somatic cells per focal plane. Data are shown as mean ± SEM (n = 5; n refers to five images from the corresponding five embryos analyzed for each genotype or condition). Two-tailed, unpaired t test results: *P < 0.05, **P < 0.01, ***P < 0.001. (G) Quantification of the mean fluorescence intensity of somatic poly(A) mRNA puncta. Data are shown as mean ± SEM (n = 23, 14, and 37 puncta for WT embryos at 26°C, atg-3 mutant embryos at 20, and 26°C, respectively; n refers to the number of puncta analyzed for each genotype or condition). Two-tailed, unpaired t test results: ***P < 0.001. (H) GTBP-1::GFP forms no distinct granules, while PGL granules accumulate in WT embryos at the ∼100–200 cell stage at 26°C. (I and J) P bodies, detected by anti-DCAP-1, are separate from PGL granules in WT embryos at the ∼100–200 cell stage at 26°C. The germ precursor cells (Z2 and Z3) are highlighted in dashed circles. J shows quantification of the colocalization of DCAP-1 bodies with PGL granules. Data are shown as mean ± SEM (n = 3; n refers to three images from the corresponding three embryos analyzed). Scale bars: 5 µm for A–D, H, and I; 2 µm for enlarged images in A–D, H, and I.

Figure S2.
Figure S2.

PGL granules act as sites for mRNA metabolism that are distinct from stress granules, related to Fig. 2. (A) The SG marker GTBP-1::GFP is diffuse in the cytoplasm in somatic cells in atg-3 mutant embryos at 26°C. (B) Numerous GTBP-1::GFP stress granules accumulate, while no PGL granules accumulate in WT embryos after heat shock treatment (33°C for 1 h). (C) GTBP-1::GFP forms numerous granules, which are separate from PGL granules in atg-3 mutant embryos after heat shock treatment (33°C for 1 h). Embryos at the ∼100–200 cell stage are shown in A–C. (D–J) The dynamics of PGL granules, shown by GFP::PGL-3, at the indicated time points after WT or atg-3 mutant embryos were shifted from 26 to 20°C. Quantification of the number of PGL granules in D–I is shown in J. 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, *P < 0.05, **P < 0.01, ***P < 0.001. Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in D–I. (K–S) The dynamics of GTBP-1::GFP granules at the indicated time points in WT embryos, atg-3 mutant embryos and pgl-3(RNAi) embryos after the embryos were shifted from 33 to 20°C. S shows the quantification of the number of GTBP-1::GFP granules in K–R. Comma-stage embryos are shown in K–R. Data are shown as mean ± SEM (n = 3; n refers to the number of embryos analyzed for each genotype or condition). Two-tailed, unpaired t test results: n.s.: no significant difference, **P < 0.01. (T) gtbp-1(RNAi) has no effect on the accumulation of PGL granules in embryos laid at 26°C. Maximum-intensity projection of Z-stack confocal images of a comma-stage embryo is shown. Scale bars: 5 µm for A–C, D–I, K–R, and T; 2 µm for enlarged images in A–C.

Figure 3.
Figure 3.

Loss of function of factors involved in mRNA metabolism suppresses the accumulation of PGL granules in embryos laid under heat stress conditions. (A) Summary of RNAi inactivations that suppress PGL granule accumulation in embryos under heat stress. (B–E) Compared to control(RNAi) (B), the number of PGL granules, labeled by GFP::PGL-3, is decreased in snr-4(RNAi) (C), ruvb-1(RNAi) (D), and pab-1(RNAi) (E) embryos at 26°C. Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in B–E. (F and G) Quantification of PGL granules per embryo in the indicated genetic backgrounds 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 results: n.s.: no significant difference, **P < 0.01, ***P < 0.001. Scale bars: 5 µm for B–E.

Figure S3.
Figure S3.

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.

Figure 4.
Figure 4.

Loss of function of factors involved in mRNA processing, transport, and translation reduces the levels of mRNA and IFE-1 partitioning into PGL granules. (A–D) Compared with control(RNAi)-treated atg-3 mutant embryos (A), the number of poly(A) mRNA-positive puncta and also the ratio of PGL granules positive for poly(A) mRNA in somatic cells is significantly decreased in atg-3 mutant embryos with simultaneous ruvb-1(RNAi) (B) and pab-1(RNAi) (C) at 26°C. Embryos at the ∼100–200 cell stage are shown in A–C. The germ precursor cells (Z2, Z3) are highlighted in dashed circles. (D and E) Quantification of the number of somatic poly(A) mRNA puncta (D) and % of PGL granules positive for poly(A) mRNA per focal plane in the indicated genotypes at 26°C (E). 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 < 0.001. (F–J) IFE-1::GFP granules are less in number and weaker in intensity in pab-1(RNAi) (G) and pab-1(RNAi); atg-3(bp412) (I) embryos at 26°C, compared with control(RNAi)-treated WT embryos (F) and atg-3(bp412) embryos (H), respectively. J shows the quantification of the number of IFE-1::GFP granules in the indicated genotypes. 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: **P < 0.01, ***P < 0.001. Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in F–I. Scale bars: 5 µm for A–C and F–I; 2 µm for enlarged images in A–C.

Figure 5.
Figure 5.

Loss of function of factors involved in mRNA turnover restores PGL granule accumulation in sept-6 mutants. (A) Summary of identified RNAi inactivations that restore PGL granule accumulation in sept-6 mutants under heat stress conditions. (B–E) Compared with control(RNAi)-treated sept-6(tm6608) mutant embryos at 26°C (B), many more PGL granules, labeled by GFP::SEPA-1, are formed in sept-6 mutant embryos treated with ntl-11(RNAi) (C), xrn-1(RNAi) (D), and eri-6(RNAi) (E). (F) Quantification of the number of PGL granules per embryo in the indicated genetic backgrounds 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 results: *P < 0.05, **P < 0.01, ***P < 0.001. (G–I) Compared to sept-6(tm6608) mutant embryos (G), many more PGL granules, detected by anti-PGL-3, are formed in dcr-1(bp132); sept-6(tm6608) mutant embryos (H) at 26°C. I shows the quantitative data (mean ± SEM, n = 3; n refers to the number of embryos analyzed for each genotype). Two-tailed, unpaired t test result: **P < 0.01. Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in B–E, G, and H. (J) The protein level of SEPA-1 is increased in extracts of dcr-1(bp132); sept-6(tm6608) mutant embryos compared with sept-6(tm6608) embryos at 26°C. Levels of SEPA-1 are normalized with the corresponding ACTIN level. Scale bars: 5 µm for B–E, G, and H. Source data are available for this figure: SourceData F5.

Figure 6.
Figure 6.

mRNA promotes LLPS and modulates the material properties of PGL condensates. (A and B) Alexa 488-labeled C. elegans total mRNAs (mRNA-Alexa 488) are partitioned into PGL condensates formed by purified PGL-1, PGL-3::mCherry, and SEPA-1 proteins (3 µM for each). More RNAs are detected in condensates when 30 ng/μl mRNA is added into the reaction compared with 10 ng/μl mRNA. (C–E) Adding mRNA enlarges the size of PGL-1/-3/SEPA-1 condensates. DIC images show condensates formed by 3 µM PGL-1/-3/SEPA-1 with no mRNA (C), 30 ng/μl mRNA (D), or 30 ng/μl mRNA pretreated with 0.5 µg/ml RNase A (E). (F) Column scatter charts of the diameters of condensates shown in C–E. Data are shown as mean ± SEM of condensates combined from three fields (220 × 166 µm) for each reaction (n = 142 for no mRNA, 121 for 10 ng/μl mRNA, and 130 for 30 ng/μl mRNA pretreated with 0.5 µg/ml RNase A; n refers to the total number of condensates analyzed for each reaction). Two-tailed, unpaired t test results: n.s.: no significant difference, ***P < 0.001. (G) Sedimentation assays showing that mRNA promotes the partitioning of PGL-1, PGL-3, and SEPA-1 proteins into condensates. S: supernatant, P: pellet. Protein levels in the pellet are normalized by the levels of the proteins in the corresponding supernatants, which is set to 1.00. (H and I) FRAP analysis of PGL-1::GFP/PGL-3/SEPA-1 condensates and PGL-1::GFP/PGL-3/SEPA-1/mRNA condensates. (I) shows quantitative FRAP data presented as mean ± SEM (n = 5; n refers to the number of bleached condensates for each reaction). (J) Adding 30 ng/μl mRNA decreases the time for two PGL-1/PGL-3::mCherry/SEPA-1 condensates to relax into a larger spherical one. The time refers to imaging time. The time for fusion (from the encounter of two condensates until complete relaxation into a larger spherical condensate) is shown as mean ± SEM (n = 10; n refers to the number of fusion events analyzed for each reaction). Two-tailed, unpaired t test results: ***P < 0.001. Scale bars: 5 µm for A and B and enlarged images in C–E; 2 µm for H and J and enlarged images in A and B; 20 µm for C–E. Source data are available for this figure: SourceData F6.

Figure S4.
Figure S4.

mRNA promotes LLPS and modulates the material properties of PGL condensates, related to Fig. 6. (A–C) Purified PGL-1/-3/SEPA-1 proteins at 0.2 μM for each fail to undergo LLPS, while a few small condensates are formed in the presence of 10 ng/μl mRNA. C shows the average number of condensates in A and B. Data are shown as mean ± SEM (n = 3; n refers to the number of randomly selected fields (220 × 166 µm) for each reaction). (D–F) Adding mRNA enlarges the size of condensates formed by purified PGL-1/-3/SEPA-1 proteins (1 µM for each protein). (F) shows column scatter charts of the diameters of condensates in D and E. Data are shown as mean ± SEM of condensates combined from three fields (220 × 166 µm) for each reaction (n = 106 for no mRNA and n = 154 for 10 ng/μl mRNA; n refers to the total number of condensates analyzed for each reaction). Two-tailed, unpaired t test result: ***P < 0.001. (G–I) Adding mRNA slightly increases the partitioning of TagRFP::IFE-1 into PGL-1/-3/SEPA-1 condensates (3 µM for each protein). (I) shows the mean TagRFP::IFE-1 fluorescence intensity per AU in condensates formed by 3 µM PGL-1/-3/SEPA-1/TagRFP::IFE-1 with or without the addition of 30 ng/μl mRNA. Data are shown as mean ± SEM (n = 27; n refers to the number of condensates analyzed for each reaction). Two-tailed, unpaired t test result: ***P < 0.001. (J–L) DIC images showing that addition of mRNA enlarges the size of condensates formed by PGL-1/-3/SEPA-1/GFP::EPG-2 (3 µM for each). (L) shows column scatter charts of the diameters of condensates in J and K. Data are shown as mean ± SEM of condensates combined from three fields (128 × 90 µm) for each reaction (n = 160 for no mRNA and n = 139 for 30 ng/μl mRNA; n refers to the total number of condensates analyzed for each reaction). Two-tailed, unpaired t test result: ***P < 0.001. (M) Protein gels for the inputs used in the in vitro LLPS assay (asterisks indicate the corresponding bands). Scale bars: 20 µm for A, B, D, E, G, H, J, and K; 5 µm for enlarged images in A, B, D, E, J, and K. Source data are available for this figure: SourceData FS4.

Figure 7.
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.

Figure S5.
Figure S5.

mRNA regulates the recruitment of EPG-2 to PGL granules, related to Fig. 7. (A) Sedimentation assays showing that mRNA reduces the level of GFP::EPG-2 that partitions into condensates. (B) The direct interaction between SEPA-1 and EPG-2 is not evidently affected by RNase A treatment or the addition of 30 ng/μl mRNA in the reactions in the in vitro GST pull-down assay. Asterisks indicate bands with the expected molecular mass. The level of EPG-2 pulled down by GST::SEPA-1 was set to 1.0 in control conditions. (C and D) Compared with control embryos shown in (Fig. 7 D), the colocalization ratio of EPG-2 aggregates and PGL granules, detected by anti-SEPA-1, is increased in snr-4(RNAi) (C) and prp-19(RNAi) (D) embryos at 26°C. Embryos at the ∼100-cell stage are shown in C and D. (E and F) Compared to epg-2 mutant embryos shown in (Fig. 7 J), the number of PGL granules, detected by PGL-3 antibody, is reduced by simultaneous snr-4(RNAi) (E) and pab-1(RNAi) (F) at 26°C. Maximum-intensity projections of Z-stack confocal images of comma-stage embryos are shown in E and F. Scale bars: 5 µm for C–F; 2 µm for enlarged images in C and D. Source data are available for this figure: SourceData FS5.

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