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A role for the arginine methylation of Rad9 in checkpoint control and cellular sensitivity to DNA damage.

He W, Ma X, Yang X, Zhao Y, Qiu J, Hang H - Nucleic Acids Res. (2011)

Bottom Line: In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5).The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated.In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules and Center for Computational and Systems Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
The genome stability is maintained by coordinated action of DNA repairs and checkpoints, which delay progression through the cell cycle in response to DNA damage. Rad9 is conserved from yeast to human and functions in cell cycle checkpoint controls. Here, a regulatory mechanism for Rad9 function is reported. In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5). Arginine methylation of Rad9 plays a critical role in S/M and G2/M cell cycle checkpoints. The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated. Additionally, Rad9 methylation is also required for cellular resistance to DNA damaging stresses. In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

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Related in: MedlinePlus

Knock down of PRMT5 influences the arginine methylation of hRad9. (A) PRMT5 is knocked down in HCT 116 cells transiently expressing PRMT5 ShRNA. Levels of PRMT5 and GAPDH were assayed in HCT116 cells expressing PRMT5 ShRNA or control ShRNA. (B) Knockdown of PRMT5 reduces the arginine methylation of hRad9. HCT116 cells expressing PRMT5 ShRNA or control ShRNA were transfected with pFLAG-CMV2-hRad9 and arginine methylation of hRad9 was detected in these cells. HCT116 cells expressing FL-hRad9-3RA were used as a negative control. (C) Methylation of hRad9 is DNA damage dependent. HeLa cells were mock treated or treated with 0.5 mM or 1 mM HU for 24 h. Ten percent cells were lysed as input, the rest cells were lysed and immunoprecipitated with ab412 antibody and then immunoblotted with anti-hRad9 monoclonal antibody.
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Figure 3: Knock down of PRMT5 influences the arginine methylation of hRad9. (A) PRMT5 is knocked down in HCT 116 cells transiently expressing PRMT5 ShRNA. Levels of PRMT5 and GAPDH were assayed in HCT116 cells expressing PRMT5 ShRNA or control ShRNA. (B) Knockdown of PRMT5 reduces the arginine methylation of hRad9. HCT116 cells expressing PRMT5 ShRNA or control ShRNA were transfected with pFLAG-CMV2-hRad9 and arginine methylation of hRad9 was detected in these cells. HCT116 cells expressing FL-hRad9-3RA were used as a negative control. (C) Methylation of hRad9 is DNA damage dependent. HeLa cells were mock treated or treated with 0.5 mM or 1 mM HU for 24 h. Ten percent cells were lysed as input, the rest cells were lysed and immunoprecipitated with ab412 antibody and then immunoblotted with anti-hRad9 monoclonal antibody.

Mentions: We have found that hRad9 is methylated in vivo and can be methylated by PRMT5 in vitro. To determine whether PRMT5 is the physiological enzyme methylating hRad9, we knocked down PRMT5 in HCT116 cells. Transfection of PRMT5 ShRNA reduced its protein level by ∼80% (Figure 3A), and this lower level of PRMT5 correlated with a dramatic reduction in the hRad9 methylation level (Figure 3B), suggesting that PRMT5 is the main enzyme for hRad9 methylation in cells.Figure 3.


A role for the arginine methylation of Rad9 in checkpoint control and cellular sensitivity to DNA damage.

He W, Ma X, Yang X, Zhao Y, Qiu J, Hang H - Nucleic Acids Res. (2011)

Knock down of PRMT5 influences the arginine methylation of hRad9. (A) PRMT5 is knocked down in HCT 116 cells transiently expressing PRMT5 ShRNA. Levels of PRMT5 and GAPDH were assayed in HCT116 cells expressing PRMT5 ShRNA or control ShRNA. (B) Knockdown of PRMT5 reduces the arginine methylation of hRad9. HCT116 cells expressing PRMT5 ShRNA or control ShRNA were transfected with pFLAG-CMV2-hRad9 and arginine methylation of hRad9 was detected in these cells. HCT116 cells expressing FL-hRad9-3RA were used as a negative control. (C) Methylation of hRad9 is DNA damage dependent. HeLa cells were mock treated or treated with 0.5 mM or 1 mM HU for 24 h. Ten percent cells were lysed as input, the rest cells were lysed and immunoprecipitated with ab412 antibody and then immunoblotted with anti-hRad9 monoclonal antibody.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Knock down of PRMT5 influences the arginine methylation of hRad9. (A) PRMT5 is knocked down in HCT 116 cells transiently expressing PRMT5 ShRNA. Levels of PRMT5 and GAPDH were assayed in HCT116 cells expressing PRMT5 ShRNA or control ShRNA. (B) Knockdown of PRMT5 reduces the arginine methylation of hRad9. HCT116 cells expressing PRMT5 ShRNA or control ShRNA were transfected with pFLAG-CMV2-hRad9 and arginine methylation of hRad9 was detected in these cells. HCT116 cells expressing FL-hRad9-3RA were used as a negative control. (C) Methylation of hRad9 is DNA damage dependent. HeLa cells were mock treated or treated with 0.5 mM or 1 mM HU for 24 h. Ten percent cells were lysed as input, the rest cells were lysed and immunoprecipitated with ab412 antibody and then immunoblotted with anti-hRad9 monoclonal antibody.
Mentions: We have found that hRad9 is methylated in vivo and can be methylated by PRMT5 in vitro. To determine whether PRMT5 is the physiological enzyme methylating hRad9, we knocked down PRMT5 in HCT116 cells. Transfection of PRMT5 ShRNA reduced its protein level by ∼80% (Figure 3A), and this lower level of PRMT5 correlated with a dramatic reduction in the hRad9 methylation level (Figure 3B), suggesting that PRMT5 is the main enzyme for hRad9 methylation in cells.Figure 3.

Bottom Line: In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5).The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated.In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules and Center for Computational and Systems Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

ABSTRACT
The genome stability is maintained by coordinated action of DNA repairs and checkpoints, which delay progression through the cell cycle in response to DNA damage. Rad9 is conserved from yeast to human and functions in cell cycle checkpoint controls. Here, a regulatory mechanism for Rad9 function is reported. In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5). Arginine methylation of Rad9 plays a critical role in S/M and G2/M cell cycle checkpoints. The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated. Additionally, Rad9 methylation is also required for cellular resistance to DNA damaging stresses. In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

Show MeSH
Related in: MedlinePlus