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Cohesin proteins promote ribosomal RNA production and protein translation in yeast and human cells.

Bose T, Lee KK, Lu S, Xu B, Harris B, Slaughter B, Unruh J, Garrett A, McDowell W, Box A, Li H, Peak A, Ramachandran S, Seidel C, Gerton JL - PLoS Genet. (2012)

Bottom Line: These mutations do not significantly affect chromosome segregation.We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation.Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies.

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Metabolic labeling of Roberts syndrome fibroblasts suggests protein translation and ribosomal RNA production are reduced.A. Cultured WT, ESCO2-mutation and V5- ESCO2-corrected human RBS fibroblasts were grown in F10 Ham Mixture plus 10% FBS. Cells were washed in PBS twice, switched to 3 mL Met/Cys-free Dulbecco's modied Eagle's medium containing 10 µM MG-132, a proteasome inhibitor, and pulsed with 30 µCi of 35S-methionine for different times (0, 15, 30, 60, 120, 240 min). Cells were lysed in RIPA buffer and proteins were precipitated by the addition of hot 10% TCA. After centrifugation, the precipitate was washed twice in acetone. The precipitate was dissolved in 100 µL of 1% SDS and heated at 95°C for 10 min. An aliquot of the SDS extract was counted in Esoscint for 35S radioactivity in a liquid scintillation spectrometer to determine the amount of 35S-methionine incorporated into proteins. B. Cultured WT, ESCO2-mutation and V5- ESCO2-corrected human RBS fibroblasts were grown in F10 Ham Mixture plus 10% FBS. 3H-uridine (5 µCi) was incubated with 106 cells from each group for two hours. Total RNA was isolated with TriZol reagent (Invitrogen, U.S.A) and the concentration of each RNA sample was measured by OD260/280. 1 µg of each sample was counted in a Beckman LS 6500 multipurpose scintillation counter to determine the amount of 3H-uridine incorporated. Four independent cultures were labeled to derive the standard deviation. Significance relative to WT was calculated using an unpaired t test. C. Ribosome profiling and quantification were carried out as described in Figure 3C.
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pgen-1002749-g007: Metabolic labeling of Roberts syndrome fibroblasts suggests protein translation and ribosomal RNA production are reduced.A. Cultured WT, ESCO2-mutation and V5- ESCO2-corrected human RBS fibroblasts were grown in F10 Ham Mixture plus 10% FBS. Cells were washed in PBS twice, switched to 3 mL Met/Cys-free Dulbecco's modied Eagle's medium containing 10 µM MG-132, a proteasome inhibitor, and pulsed with 30 µCi of 35S-methionine for different times (0, 15, 30, 60, 120, 240 min). Cells were lysed in RIPA buffer and proteins were precipitated by the addition of hot 10% TCA. After centrifugation, the precipitate was washed twice in acetone. The precipitate was dissolved in 100 µL of 1% SDS and heated at 95°C for 10 min. An aliquot of the SDS extract was counted in Esoscint for 35S radioactivity in a liquid scintillation spectrometer to determine the amount of 35S-methionine incorporated into proteins. B. Cultured WT, ESCO2-mutation and V5- ESCO2-corrected human RBS fibroblasts were grown in F10 Ham Mixture plus 10% FBS. 3H-uridine (5 µCi) was incubated with 106 cells from each group for two hours. Total RNA was isolated with TriZol reagent (Invitrogen, U.S.A) and the concentration of each RNA sample was measured by OD260/280. 1 µg of each sample was counted in a Beckman LS 6500 multipurpose scintillation counter to determine the amount of 3H-uridine incorporated. Four independent cultures were labeled to derive the standard deviation. Significance relative to WT was calculated using an unpaired t test. C. Ribosome profiling and quantification were carried out as described in Figure 3C.

Mentions: Given that the eco1-W216G mutation is associated with reduced protein translation and rRNA production in budding yeast, we decided to investigate whether a human cell line bearing the same mutation displays similar physiology. We used 35S-methionine labeling to measure protein synthesis in 1) a Roberts syndrome fibroblast cell line, 2) a version of the cell line that has been corrected with a wild-type copy of ESCO2 [45] and 3) a normal fibroblast line. We found that the Roberts syndrome cells incorporated methionine at about 50% the level as the corrected line or a normal fibroblast line (Figure 7A), very similar to the observations in yeast (Figure 3D). Furthermore, we measured the incorporation of 3H-uridine as an indicator of ribosomal RNA synthesis. We found that the rate of incorporation in the Roberts syndrome cells is about 50% the level as the corrected line or a normal fibroblast line (Figure 7B), very similar to the observation in yeast (Figure 5B). Finally, we examined the polysome profile in the Roberts cells. We find that the polysome to 80S ratio is lower in the Roberts cells relative to the corrected line (Figure 7C), similar to the observation in yeast (Figure 3C). Thus, it appears that protein synthesis and ribosomal RNA production are reduced in human Roberts syndrome fibroblasts, and suggests that the findings in yeast are relevant to the human disease.


Cohesin proteins promote ribosomal RNA production and protein translation in yeast and human cells.

Bose T, Lee KK, Lu S, Xu B, Harris B, Slaughter B, Unruh J, Garrett A, McDowell W, Box A, Li H, Peak A, Ramachandran S, Seidel C, Gerton JL - PLoS Genet. (2012)

Metabolic labeling of Roberts syndrome fibroblasts suggests protein translation and ribosomal RNA production are reduced.A. Cultured WT, ESCO2-mutation and V5- ESCO2-corrected human RBS fibroblasts were grown in F10 Ham Mixture plus 10% FBS. Cells were washed in PBS twice, switched to 3 mL Met/Cys-free Dulbecco's modied Eagle's medium containing 10 µM MG-132, a proteasome inhibitor, and pulsed with 30 µCi of 35S-methionine for different times (0, 15, 30, 60, 120, 240 min). Cells were lysed in RIPA buffer and proteins were precipitated by the addition of hot 10% TCA. After centrifugation, the precipitate was washed twice in acetone. The precipitate was dissolved in 100 µL of 1% SDS and heated at 95°C for 10 min. An aliquot of the SDS extract was counted in Esoscint for 35S radioactivity in a liquid scintillation spectrometer to determine the amount of 35S-methionine incorporated into proteins. B. Cultured WT, ESCO2-mutation and V5- ESCO2-corrected human RBS fibroblasts were grown in F10 Ham Mixture plus 10% FBS. 3H-uridine (5 µCi) was incubated with 106 cells from each group for two hours. Total RNA was isolated with TriZol reagent (Invitrogen, U.S.A) and the concentration of each RNA sample was measured by OD260/280. 1 µg of each sample was counted in a Beckman LS 6500 multipurpose scintillation counter to determine the amount of 3H-uridine incorporated. Four independent cultures were labeled to derive the standard deviation. Significance relative to WT was calculated using an unpaired t test. C. Ribosome profiling and quantification were carried out as described in Figure 3C.
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pgen-1002749-g007: Metabolic labeling of Roberts syndrome fibroblasts suggests protein translation and ribosomal RNA production are reduced.A. Cultured WT, ESCO2-mutation and V5- ESCO2-corrected human RBS fibroblasts were grown in F10 Ham Mixture plus 10% FBS. Cells were washed in PBS twice, switched to 3 mL Met/Cys-free Dulbecco's modied Eagle's medium containing 10 µM MG-132, a proteasome inhibitor, and pulsed with 30 µCi of 35S-methionine for different times (0, 15, 30, 60, 120, 240 min). Cells were lysed in RIPA buffer and proteins were precipitated by the addition of hot 10% TCA. After centrifugation, the precipitate was washed twice in acetone. The precipitate was dissolved in 100 µL of 1% SDS and heated at 95°C for 10 min. An aliquot of the SDS extract was counted in Esoscint for 35S radioactivity in a liquid scintillation spectrometer to determine the amount of 35S-methionine incorporated into proteins. B. Cultured WT, ESCO2-mutation and V5- ESCO2-corrected human RBS fibroblasts were grown in F10 Ham Mixture plus 10% FBS. 3H-uridine (5 µCi) was incubated with 106 cells from each group for two hours. Total RNA was isolated with TriZol reagent (Invitrogen, U.S.A) and the concentration of each RNA sample was measured by OD260/280. 1 µg of each sample was counted in a Beckman LS 6500 multipurpose scintillation counter to determine the amount of 3H-uridine incorporated. Four independent cultures were labeled to derive the standard deviation. Significance relative to WT was calculated using an unpaired t test. C. Ribosome profiling and quantification were carried out as described in Figure 3C.
Mentions: Given that the eco1-W216G mutation is associated with reduced protein translation and rRNA production in budding yeast, we decided to investigate whether a human cell line bearing the same mutation displays similar physiology. We used 35S-methionine labeling to measure protein synthesis in 1) a Roberts syndrome fibroblast cell line, 2) a version of the cell line that has been corrected with a wild-type copy of ESCO2 [45] and 3) a normal fibroblast line. We found that the Roberts syndrome cells incorporated methionine at about 50% the level as the corrected line or a normal fibroblast line (Figure 7A), very similar to the observations in yeast (Figure 3D). Furthermore, we measured the incorporation of 3H-uridine as an indicator of ribosomal RNA synthesis. We found that the rate of incorporation in the Roberts syndrome cells is about 50% the level as the corrected line or a normal fibroblast line (Figure 7B), very similar to the observation in yeast (Figure 5B). Finally, we examined the polysome profile in the Roberts cells. We find that the polysome to 80S ratio is lower in the Roberts cells relative to the corrected line (Figure 7C), similar to the observation in yeast (Figure 3C). Thus, it appears that protein synthesis and ribosomal RNA production are reduced in human Roberts syndrome fibroblasts, and suggests that the findings in yeast are relevant to the human disease.

Bottom Line: These mutations do not significantly affect chromosome segregation.We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation.Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and (35)S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies.

Show MeSH
Related in: MedlinePlus