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Rpb1 sumoylation in response to UV radiation or transcriptional impairment in yeast.

Chen X, Ding B, LeJeune D, Ruggiero C, Li S - PLoS ONE (2009)

Bottom Line: K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site.Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk.However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.

ABSTRACT
Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II (Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1 is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an important link between the modification and the DNA damage checkpoint response.

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Western blots showing the roles of Ubc9 and Siz1 in UV-induced Rpb1 sumoylation.(A) Degradation of degron-myc tagged Ubc9 upon shifting to nonpermissive temperature (37°C) in galactose containing medium (to induce the expression of plasmid pKL142 encoded Ubr1, a ubiquitin E3 ligase). Tubulin serves as an internal loading control. (B) Abolishment of UV-induced Rpb1 sumoylation when Ubc9 was depleted. Rpb1 was immunoprecipitated from the cells cultured at the indicated conditions using antibody 8WG16 and probed with anti-SUMO and 8WG16 antibodies. (C) The roles of Siz1 and Siz2 in UV-induced Rpb1 sumoylation. Rpb1 was immunoprecipited from the UV irradiated wild type (BY4741) and mutant (strains 4245 and 2412) cells using antibody 8WG16 and probed with anti-SUMO and 8WG16 antibodies. The control was a sample prepared from unirradiated wild type cells. WT, wild type.
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pone-0005267-g002: Western blots showing the roles of Ubc9 and Siz1 in UV-induced Rpb1 sumoylation.(A) Degradation of degron-myc tagged Ubc9 upon shifting to nonpermissive temperature (37°C) in galactose containing medium (to induce the expression of plasmid pKL142 encoded Ubr1, a ubiquitin E3 ligase). Tubulin serves as an internal loading control. (B) Abolishment of UV-induced Rpb1 sumoylation when Ubc9 was depleted. Rpb1 was immunoprecipitated from the cells cultured at the indicated conditions using antibody 8WG16 and probed with anti-SUMO and 8WG16 antibodies. (C) The roles of Siz1 and Siz2 in UV-induced Rpb1 sumoylation. Rpb1 was immunoprecipited from the UV irradiated wild type (BY4741) and mutant (strains 4245 and 2412) cells using antibody 8WG16 and probed with anti-SUMO and 8WG16 antibodies. The control was a sample prepared from unirradiated wild type cells. WT, wild type.

Mentions: Ubc9, which is essential for cell viability, is the only SUMO E2 conjugase identified so far in yeast [23]. To examine if Ubc9 is involved in Rpb1 sumoylation, we inserted the degron-myc sequences [38], [39] in-frame at the 5′ end of the coding region of the genomic UBC9 gene, thereby expressing the Ubc9 protein with a degron-myc tag at the N-terminus. Degron tagging has been successfully used to conditionally degrade an essential protein in yeast [38], [39]. A degron tagged protein can function normally in cells at permissive temperature (24°C), but is rapidly degraded through a ubiquitin-mediated pathway at non-permissive temperature (37°C) [39]. The myc tag following the degron tag is used for detection of a tagged protein with a generic anti-myc antibody [38], [39]. The tagged Ubc9 was degraded to an undetectable level ∼2.5 hours after the cells were shifted to 37°C (Fig. 2A). In cells expressing the degron-myc tagged Ubc9, UV induced Rpb1 sumoylation occurred normally at the permissive temperature but was undetectable at the non-permissive temperature (Fig. 2B). On the other hand, Rpb1 sumoylation occurred normally in cells expressing the native Ubc9 at the nonpermissive temperature. These results indicate that the SUMO E2 conjugase Ubc9 plays an important role in Rpb1 sumoylation.


Rpb1 sumoylation in response to UV radiation or transcriptional impairment in yeast.

Chen X, Ding B, LeJeune D, Ruggiero C, Li S - PLoS ONE (2009)

Western blots showing the roles of Ubc9 and Siz1 in UV-induced Rpb1 sumoylation.(A) Degradation of degron-myc tagged Ubc9 upon shifting to nonpermissive temperature (37°C) in galactose containing medium (to induce the expression of plasmid pKL142 encoded Ubr1, a ubiquitin E3 ligase). Tubulin serves as an internal loading control. (B) Abolishment of UV-induced Rpb1 sumoylation when Ubc9 was depleted. Rpb1 was immunoprecipitated from the cells cultured at the indicated conditions using antibody 8WG16 and probed with anti-SUMO and 8WG16 antibodies. (C) The roles of Siz1 and Siz2 in UV-induced Rpb1 sumoylation. Rpb1 was immunoprecipited from the UV irradiated wild type (BY4741) and mutant (strains 4245 and 2412) cells using antibody 8WG16 and probed with anti-SUMO and 8WG16 antibodies. The control was a sample prepared from unirradiated wild type cells. WT, wild type.
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getmorefigures.php?uid=PMC2668072&req=5

pone-0005267-g002: Western blots showing the roles of Ubc9 and Siz1 in UV-induced Rpb1 sumoylation.(A) Degradation of degron-myc tagged Ubc9 upon shifting to nonpermissive temperature (37°C) in galactose containing medium (to induce the expression of plasmid pKL142 encoded Ubr1, a ubiquitin E3 ligase). Tubulin serves as an internal loading control. (B) Abolishment of UV-induced Rpb1 sumoylation when Ubc9 was depleted. Rpb1 was immunoprecipitated from the cells cultured at the indicated conditions using antibody 8WG16 and probed with anti-SUMO and 8WG16 antibodies. (C) The roles of Siz1 and Siz2 in UV-induced Rpb1 sumoylation. Rpb1 was immunoprecipited from the UV irradiated wild type (BY4741) and mutant (strains 4245 and 2412) cells using antibody 8WG16 and probed with anti-SUMO and 8WG16 antibodies. The control was a sample prepared from unirradiated wild type cells. WT, wild type.
Mentions: Ubc9, which is essential for cell viability, is the only SUMO E2 conjugase identified so far in yeast [23]. To examine if Ubc9 is involved in Rpb1 sumoylation, we inserted the degron-myc sequences [38], [39] in-frame at the 5′ end of the coding region of the genomic UBC9 gene, thereby expressing the Ubc9 protein with a degron-myc tag at the N-terminus. Degron tagging has been successfully used to conditionally degrade an essential protein in yeast [38], [39]. A degron tagged protein can function normally in cells at permissive temperature (24°C), but is rapidly degraded through a ubiquitin-mediated pathway at non-permissive temperature (37°C) [39]. The myc tag following the degron tag is used for detection of a tagged protein with a generic anti-myc antibody [38], [39]. The tagged Ubc9 was degraded to an undetectable level ∼2.5 hours after the cells were shifted to 37°C (Fig. 2A). In cells expressing the degron-myc tagged Ubc9, UV induced Rpb1 sumoylation occurred normally at the permissive temperature but was undetectable at the non-permissive temperature (Fig. 2B). On the other hand, Rpb1 sumoylation occurred normally in cells expressing the native Ubc9 at the nonpermissive temperature. These results indicate that the SUMO E2 conjugase Ubc9 plays an important role in Rpb1 sumoylation.

Bottom Line: K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site.Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk.However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.

ABSTRACT
Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II (Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1 is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an important link between the modification and the DNA damage checkpoint response.

Show MeSH
Related in: MedlinePlus