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Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules.

Bounedjah O, Desforges B, Wu TD, Pioche-Durieu C, Marco S, Hamon L, Curmi PA, Guerquin-Kern JL, Piétrement O, Pastré D - Nucleic Acids Res. (2014)

Bottom Line: We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly.Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly.Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), UMR829; Université Evry-Val d'Essonne, Evry 91025, France.

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Related in: MedlinePlus

Stress granules in arsenite-treated cells are enriched in RNA compared to proteins. (A) NRK cells were untreated or exposed to 300 μM arsenite for 45 min. Transmission electron microscopy shows the presence of polysomes in control condition (higher magnification), which were not present after arsenite treatment. Instead, large and unstructured aggregates presumed to be stress granules appear in the cytoplasm. (B) Transmission electron microscopy imaging of immunogold anti-YB-1 labeled NRK cells shows a concentration of gold nanoparticles in the granules that appeared with arsenite. Statistical analysis of the quantification of the gold particle location in the cytoplasm of control and arsenite-treated cells and in the granules indicates an about 4-times enrichment of gold nanoparticles in the granules. Results are mean ± SD (n = 30). **P < 0.01 by t-test. (C) Correlative TEM/nanoSIMS microscopy of NRK cells treated with arsenite. The15N:14N ratio highlights the15N-enriched uridine-labeled RNA. Dashed circles and arrows point out a RNA-rich stress granule and a nucleolus, respectively. (D) nanoSIMS mapping of the 12C14N− and 12C15N− ion species and of the 15N:14N ratio in control and arsenite-treated cells (see ‘Materials and Methods’ section). In arsenite-treated cells, we observed the formation of stress granules (dashed circles) which can be distinguished both in the 12C14N− (proteins and RNA) and the 12C15N− (15N-uridine-labeled RNA) cartographies. As a control, arrows indicate the presence of RNA-rich nucleolus in both control and arsenite-treated cells. High magnification images of cytoplasmic stress granules show their enrichment in RNA compared to proteins as evidenced in the 15N:14N cartography. Scale bars: 5 μm. (E) Image gallery of stress granules and their respective line profile illustrating the increase of the15N:14N ratio in stress granules (mean value obtained over the 230 nm-thick dashed line). The scatter plots represent the mean 15N:14N ratio values measured in the cytoplasm of control and arsenite-treated cells (outside and inside stress granules in the latter case). The means were obtained by averaging the ratio over 0.5 μm2 areas. Statistical results are mean ± SD (n = 50). **P < 0.01 by t-test.
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Figure 1: Stress granules in arsenite-treated cells are enriched in RNA compared to proteins. (A) NRK cells were untreated or exposed to 300 μM arsenite for 45 min. Transmission electron microscopy shows the presence of polysomes in control condition (higher magnification), which were not present after arsenite treatment. Instead, large and unstructured aggregates presumed to be stress granules appear in the cytoplasm. (B) Transmission electron microscopy imaging of immunogold anti-YB-1 labeled NRK cells shows a concentration of gold nanoparticles in the granules that appeared with arsenite. Statistical analysis of the quantification of the gold particle location in the cytoplasm of control and arsenite-treated cells and in the granules indicates an about 4-times enrichment of gold nanoparticles in the granules. Results are mean ± SD (n = 30). **P < 0.01 by t-test. (C) Correlative TEM/nanoSIMS microscopy of NRK cells treated with arsenite. The15N:14N ratio highlights the15N-enriched uridine-labeled RNA. Dashed circles and arrows point out a RNA-rich stress granule and a nucleolus, respectively. (D) nanoSIMS mapping of the 12C14N− and 12C15N− ion species and of the 15N:14N ratio in control and arsenite-treated cells (see ‘Materials and Methods’ section). In arsenite-treated cells, we observed the formation of stress granules (dashed circles) which can be distinguished both in the 12C14N− (proteins and RNA) and the 12C15N− (15N-uridine-labeled RNA) cartographies. As a control, arrows indicate the presence of RNA-rich nucleolus in both control and arsenite-treated cells. High magnification images of cytoplasmic stress granules show their enrichment in RNA compared to proteins as evidenced in the 15N:14N cartography. Scale bars: 5 μm. (E) Image gallery of stress granules and their respective line profile illustrating the increase of the15N:14N ratio in stress granules (mean value obtained over the 230 nm-thick dashed line). The scatter plots represent the mean 15N:14N ratio values measured in the cytoplasm of control and arsenite-treated cells (outside and inside stress granules in the latter case). The means were obtained by averaging the ratio over 0.5 μm2 areas. Statistical results are mean ± SD (n = 50). **P < 0.01 by t-test.

Mentions: Mapping of the 15N:14N ratio showed an uniform distribution over a 9 μm2 area and statistical analysis of this ratio indicates that the RNA sample has a significant higher 15N content (15N:14N ratio = 3.96 ± 0.04%) compared to the cytoplasm of NRK cells exposed to 15N-uridine (15N:14N ratio = 0.79 ± 0.23%), see Figure 1E. This result thus indicates that 15N-uridine has been significantly incorporated to cellular RNA which is necessary to keep a high signal-to-noise ratio in the 15N:14N cartography.


Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules.

Bounedjah O, Desforges B, Wu TD, Pioche-Durieu C, Marco S, Hamon L, Curmi PA, Guerquin-Kern JL, Piétrement O, Pastré D - Nucleic Acids Res. (2014)

Stress granules in arsenite-treated cells are enriched in RNA compared to proteins. (A) NRK cells were untreated or exposed to 300 μM arsenite for 45 min. Transmission electron microscopy shows the presence of polysomes in control condition (higher magnification), which were not present after arsenite treatment. Instead, large and unstructured aggregates presumed to be stress granules appear in the cytoplasm. (B) Transmission electron microscopy imaging of immunogold anti-YB-1 labeled NRK cells shows a concentration of gold nanoparticles in the granules that appeared with arsenite. Statistical analysis of the quantification of the gold particle location in the cytoplasm of control and arsenite-treated cells and in the granules indicates an about 4-times enrichment of gold nanoparticles in the granules. Results are mean ± SD (n = 30). **P < 0.01 by t-test. (C) Correlative TEM/nanoSIMS microscopy of NRK cells treated with arsenite. The15N:14N ratio highlights the15N-enriched uridine-labeled RNA. Dashed circles and arrows point out a RNA-rich stress granule and a nucleolus, respectively. (D) nanoSIMS mapping of the 12C14N− and 12C15N− ion species and of the 15N:14N ratio in control and arsenite-treated cells (see ‘Materials and Methods’ section). In arsenite-treated cells, we observed the formation of stress granules (dashed circles) which can be distinguished both in the 12C14N− (proteins and RNA) and the 12C15N− (15N-uridine-labeled RNA) cartographies. As a control, arrows indicate the presence of RNA-rich nucleolus in both control and arsenite-treated cells. High magnification images of cytoplasmic stress granules show their enrichment in RNA compared to proteins as evidenced in the 15N:14N cartography. Scale bars: 5 μm. (E) Image gallery of stress granules and their respective line profile illustrating the increase of the15N:14N ratio in stress granules (mean value obtained over the 230 nm-thick dashed line). The scatter plots represent the mean 15N:14N ratio values measured in the cytoplasm of control and arsenite-treated cells (outside and inside stress granules in the latter case). The means were obtained by averaging the ratio over 0.5 μm2 areas. Statistical results are mean ± SD (n = 50). **P < 0.01 by t-test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Stress granules in arsenite-treated cells are enriched in RNA compared to proteins. (A) NRK cells were untreated or exposed to 300 μM arsenite for 45 min. Transmission electron microscopy shows the presence of polysomes in control condition (higher magnification), which were not present after arsenite treatment. Instead, large and unstructured aggregates presumed to be stress granules appear in the cytoplasm. (B) Transmission electron microscopy imaging of immunogold anti-YB-1 labeled NRK cells shows a concentration of gold nanoparticles in the granules that appeared with arsenite. Statistical analysis of the quantification of the gold particle location in the cytoplasm of control and arsenite-treated cells and in the granules indicates an about 4-times enrichment of gold nanoparticles in the granules. Results are mean ± SD (n = 30). **P < 0.01 by t-test. (C) Correlative TEM/nanoSIMS microscopy of NRK cells treated with arsenite. The15N:14N ratio highlights the15N-enriched uridine-labeled RNA. Dashed circles and arrows point out a RNA-rich stress granule and a nucleolus, respectively. (D) nanoSIMS mapping of the 12C14N− and 12C15N− ion species and of the 15N:14N ratio in control and arsenite-treated cells (see ‘Materials and Methods’ section). In arsenite-treated cells, we observed the formation of stress granules (dashed circles) which can be distinguished both in the 12C14N− (proteins and RNA) and the 12C15N− (15N-uridine-labeled RNA) cartographies. As a control, arrows indicate the presence of RNA-rich nucleolus in both control and arsenite-treated cells. High magnification images of cytoplasmic stress granules show their enrichment in RNA compared to proteins as evidenced in the 15N:14N cartography. Scale bars: 5 μm. (E) Image gallery of stress granules and their respective line profile illustrating the increase of the15N:14N ratio in stress granules (mean value obtained over the 230 nm-thick dashed line). The scatter plots represent the mean 15N:14N ratio values measured in the cytoplasm of control and arsenite-treated cells (outside and inside stress granules in the latter case). The means were obtained by averaging the ratio over 0.5 μm2 areas. Statistical results are mean ± SD (n = 50). **P < 0.01 by t-test.
Mentions: Mapping of the 15N:14N ratio showed an uniform distribution over a 9 μm2 area and statistical analysis of this ratio indicates that the RNA sample has a significant higher 15N content (15N:14N ratio = 3.96 ± 0.04%) compared to the cytoplasm of NRK cells exposed to 15N-uridine (15N:14N ratio = 0.79 ± 0.23%), see Figure 1E. This result thus indicates that 15N-uridine has been significantly incorporated to cellular RNA which is necessary to keep a high signal-to-noise ratio in the 15N:14N cartography.

Bottom Line: We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly.Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly.Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), UMR829; Université Evry-Val d'Essonne, Evry 91025, France.

Show MeSH
Related in: MedlinePlus