Limits...
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.

Show MeSH

Related in: MedlinePlus

Cohesin mutations compromise production of ribosomal RNA.A. The ratios for microarray probes corresponding to the 25S, 5.8S and 18S transcripts of the rDNA locus are shown for eco1-W216G/WT and scc2-D730V/WT at time 0 from Figure 1. The x axis corresponds to SGD coordinates, ordered by the beginning of the probe with the midpoint of the probe given. The arrow indicates the direction of transcription. The error bars show the standard error. B. Strains were in log phase in SD-ura at 30°C when an aliquot was removed and 3H-uridine was added for 5 min to equal numbers of cells for each strain background. Incorporation was measured by scintillation counting after extensive washing of the cells. Three independent cultures were labeled to derive the standard deviation. Significance was calculated using an unpaired t test. C. A growth curve is shown for the strains in SD-ura medium at 30°C. A similar experiment was performed in the W303a background and is included in Figure S4. D. Strains were grown in SD-met at 30°C and RNA was extracted from equal numbers of cells following a 5 minute pulse with 3H-methylmethionine and a chase with cold methionine. Equal amounts of RNA were run on a formaldehyde gel and photographed following staining with ethidium bromide (EtBr). Then the RNA was transferred to a membrane for exposure. Following exposure, the bands were excised and radioactivity was determined by scintillation counting. Percent incorporation is given as a fraction of WT. Independent biological replicates are shown. E. A growth curve is shown for the strains in SD-met medium at 30°C. F. Growth of the eco1-W216G mutant at 33°C is partially rescued by expression of the rDNA from a Pol II (gal) promoter. G. Total RNA was isolated from the strains shown following growth in raffinose followed by a 5.5. hour incubation with either glucose or galactose. The total amount of 28S+18S was quantified in glucose and galactose.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3375231&req=5

pgen-1002749-g005: Cohesin mutations compromise production of ribosomal RNA.A. The ratios for microarray probes corresponding to the 25S, 5.8S and 18S transcripts of the rDNA locus are shown for eco1-W216G/WT and scc2-D730V/WT at time 0 from Figure 1. The x axis corresponds to SGD coordinates, ordered by the beginning of the probe with the midpoint of the probe given. The arrow indicates the direction of transcription. The error bars show the standard error. B. Strains were in log phase in SD-ura at 30°C when an aliquot was removed and 3H-uridine was added for 5 min to equal numbers of cells for each strain background. Incorporation was measured by scintillation counting after extensive washing of the cells. Three independent cultures were labeled to derive the standard deviation. Significance was calculated using an unpaired t test. C. A growth curve is shown for the strains in SD-ura medium at 30°C. A similar experiment was performed in the W303a background and is included in Figure S4. D. Strains were grown in SD-met at 30°C and RNA was extracted from equal numbers of cells following a 5 minute pulse with 3H-methylmethionine and a chase with cold methionine. Equal amounts of RNA were run on a formaldehyde gel and photographed following staining with ethidium bromide (EtBr). Then the RNA was transferred to a membrane for exposure. Following exposure, the bands were excised and radioactivity was determined by scintillation counting. Percent incorporation is given as a fraction of WT. Independent biological replicates are shown. E. A growth curve is shown for the strains in SD-met medium at 30°C. F. Growth of the eco1-W216G mutant at 33°C is partially rescued by expression of the rDNA from a Pol II (gal) promoter. G. Total RNA was isolated from the strains shown following growth in raffinose followed by a 5.5. hour incubation with either glucose or galactose. The total amount of 28S+18S was quantified in glucose and galactose.

Mentions: A. Whole cell extracts were made from a WT, scc2-D730V, smc1-Q843Δ, and eco1-W216G mutant strains grown in YPD+CSM at 30°C. Extracts were used for Western blotting to measure levels of eiF2α protein, and phospho-eiF2α, which is an indicator of translational inhibition. Biological replicates yielded similar results (the first number corresponds to the blot shown). B. Growth profiles are shown for WT, scc2-D730V, smc1-Q843Δ, and eco1-W216G mutant strains. Profiles were collected at 15 minute intervals in triplicate for each strain in YPD+CSM at 30°C; a single curve is shown. We derived the maximum slope of the curves in log phase and tested whether the slopes were significantly different for replicates of the same genotype or for WT versus mutant (for more information see Materials and Methods). None of the curves derived from a single genotype showed statistical significance between replicates. The p value for the comparison to WT is indicated where significant. C. Polysome profiles of WT, smc1-Q843Δ, and eco1-W216G mutant strains were collected from cells grown in YPD+CSM at 30°C. The ratio of polysomes to 80S (P/80S) is shown. Profiling was conducted at least twice with similar results. Quantification was carried out using Mathematica and Image J software with similar results. Results from Image J analysis are shown. D. Strains growing in log phase in SD-met+35S-methionine at 30°C (see Figure 5E for growth profile) were used to measure protein synthesis. We verified that the cohesin mutants are not methionine auxotrophs. E. WT and eco1-W216G mutant strains with the Gcn4-lacZ transgene integrated at the TRP1 locus were transformed with either empty vector (EV) or a plasmid constitutively overexpressing the ternary complex (TC) by virtue of its high copy. Strains were grown and assayed as described in Figure 2. The difference between eco1-W216G+EV and eco1-W216G+TC was significant at p<0.0001. See Figure S3 for verification that the smc1-Q843Δ and eco1-W216G strains used throughout the manuscript are not aneuploid.


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)

Cohesin mutations compromise production of ribosomal RNA.A. The ratios for microarray probes corresponding to the 25S, 5.8S and 18S transcripts of the rDNA locus are shown for eco1-W216G/WT and scc2-D730V/WT at time 0 from Figure 1. The x axis corresponds to SGD coordinates, ordered by the beginning of the probe with the midpoint of the probe given. The arrow indicates the direction of transcription. The error bars show the standard error. B. Strains were in log phase in SD-ura at 30°C when an aliquot was removed and 3H-uridine was added for 5 min to equal numbers of cells for each strain background. Incorporation was measured by scintillation counting after extensive washing of the cells. Three independent cultures were labeled to derive the standard deviation. Significance was calculated using an unpaired t test. C. A growth curve is shown for the strains in SD-ura medium at 30°C. A similar experiment was performed in the W303a background and is included in Figure S4. D. Strains were grown in SD-met at 30°C and RNA was extracted from equal numbers of cells following a 5 minute pulse with 3H-methylmethionine and a chase with cold methionine. Equal amounts of RNA were run on a formaldehyde gel and photographed following staining with ethidium bromide (EtBr). Then the RNA was transferred to a membrane for exposure. Following exposure, the bands were excised and radioactivity was determined by scintillation counting. Percent incorporation is given as a fraction of WT. Independent biological replicates are shown. E. A growth curve is shown for the strains in SD-met medium at 30°C. F. Growth of the eco1-W216G mutant at 33°C is partially rescued by expression of the rDNA from a Pol II (gal) promoter. G. Total RNA was isolated from the strains shown following growth in raffinose followed by a 5.5. hour incubation with either glucose or galactose. The total amount of 28S+18S was quantified in glucose and galactose.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375231&req=5

pgen-1002749-g005: Cohesin mutations compromise production of ribosomal RNA.A. The ratios for microarray probes corresponding to the 25S, 5.8S and 18S transcripts of the rDNA locus are shown for eco1-W216G/WT and scc2-D730V/WT at time 0 from Figure 1. The x axis corresponds to SGD coordinates, ordered by the beginning of the probe with the midpoint of the probe given. The arrow indicates the direction of transcription. The error bars show the standard error. B. Strains were in log phase in SD-ura at 30°C when an aliquot was removed and 3H-uridine was added for 5 min to equal numbers of cells for each strain background. Incorporation was measured by scintillation counting after extensive washing of the cells. Three independent cultures were labeled to derive the standard deviation. Significance was calculated using an unpaired t test. C. A growth curve is shown for the strains in SD-ura medium at 30°C. A similar experiment was performed in the W303a background and is included in Figure S4. D. Strains were grown in SD-met at 30°C and RNA was extracted from equal numbers of cells following a 5 minute pulse with 3H-methylmethionine and a chase with cold methionine. Equal amounts of RNA were run on a formaldehyde gel and photographed following staining with ethidium bromide (EtBr). Then the RNA was transferred to a membrane for exposure. Following exposure, the bands were excised and radioactivity was determined by scintillation counting. Percent incorporation is given as a fraction of WT. Independent biological replicates are shown. E. A growth curve is shown for the strains in SD-met medium at 30°C. F. Growth of the eco1-W216G mutant at 33°C is partially rescued by expression of the rDNA from a Pol II (gal) promoter. G. Total RNA was isolated from the strains shown following growth in raffinose followed by a 5.5. hour incubation with either glucose or galactose. The total amount of 28S+18S was quantified in glucose and galactose.
Mentions: A. Whole cell extracts were made from a WT, scc2-D730V, smc1-Q843Δ, and eco1-W216G mutant strains grown in YPD+CSM at 30°C. Extracts were used for Western blotting to measure levels of eiF2α protein, and phospho-eiF2α, which is an indicator of translational inhibition. Biological replicates yielded similar results (the first number corresponds to the blot shown). B. Growth profiles are shown for WT, scc2-D730V, smc1-Q843Δ, and eco1-W216G mutant strains. Profiles were collected at 15 minute intervals in triplicate for each strain in YPD+CSM at 30°C; a single curve is shown. We derived the maximum slope of the curves in log phase and tested whether the slopes were significantly different for replicates of the same genotype or for WT versus mutant (for more information see Materials and Methods). None of the curves derived from a single genotype showed statistical significance between replicates. The p value for the comparison to WT is indicated where significant. C. Polysome profiles of WT, smc1-Q843Δ, and eco1-W216G mutant strains were collected from cells grown in YPD+CSM at 30°C. The ratio of polysomes to 80S (P/80S) is shown. Profiling was conducted at least twice with similar results. Quantification was carried out using Mathematica and Image J software with similar results. Results from Image J analysis are shown. D. Strains growing in log phase in SD-met+35S-methionine at 30°C (see Figure 5E for growth profile) were used to measure protein synthesis. We verified that the cohesin mutants are not methionine auxotrophs. E. WT and eco1-W216G mutant strains with the Gcn4-lacZ transgene integrated at the TRP1 locus were transformed with either empty vector (EV) or a plasmid constitutively overexpressing the ternary complex (TC) by virtue of its high copy. Strains were grown and assayed as described in Figure 2. The difference between eco1-W216G+EV and eco1-W216G+TC was significant at p<0.0001. See Figure S3 for verification that the smc1-Q843Δ and eco1-W216G strains used throughout the manuscript are not aneuploid.

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