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.

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.

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