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A new connection of mRNP biogenesis and export with transcription-coupled repair.

Gaillard H, Wellinger RE, Aguilera A - Nucleic Acids Res. (2007)

Bottom Line: Careful analysis revealed that THO mutants are also specifically affected in TCR.Along with severe UV damage-dependent loss in processivity, RNAPII was found binding to chromatin upon UV irradiation in THO mutants, suggesting that RNAPII remains stalled at DNA lesions.Our results indicate that RNAPII is not proficient for TCR in mRNP biogenesis and export mutants, opening new perspectives on our knowledge of TCR in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Facultad de Biología, Universidad de Sevilla, CABIMER, CSIC-Universidad de Sevilla, Avenida Américo Vespucio s/n, 41092 Sevilla, Spain.

ABSTRACT
Although DNA repair is faster in the transcribed strand of active genes, little is known about the possible contribution of mRNP biogenesis and export in transcription-coupled repair (TCR). Interestingly, mutants of THO, a transcription complex involved in maintenance of genome integrity, mRNP biogenesis and export, were recently found to be deficient in nucleotide excision repair. In this study we show by molecular DNA repair analysis, that Sub2-Yra1 and Thp1-Sac3, two main mRNA export complexes, are required for efficient TCR in yeast. Careful analysis revealed that THO mutants are also specifically affected in TCR. Ribozyme-mediated mRNA self-cleavage between two hot spots for UV damage showed that efficient TCR does not depend on the nascent mRNA, neither in wild-type nor in mutant cells. Along with severe UV damage-dependent loss in processivity, RNAPII was found binding to chromatin upon UV irradiation in THO mutants, suggesting that RNAPII remains stalled at DNA lesions. Furthermore, Def1, a factor responsible for the degradation of stalled RNAPII, appears essential for the viability of THO mutants subjected to DNA damage. Our results indicate that RNAPII is not proficient for TCR in mRNP biogenesis and export mutants, opening new perspectives on our knowledge of TCR in eukaryotic cells.

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mRNA export deficient cells are sensitive to UV in the absence of global genome repair. UV sensitivity curves of sub2-206, mex67-5, thp1Δ, sac3Δ, hpr1-101, and rad7Δ single and double mutants (plain symbols). Isogenic wild-type, NER-deficient rad1Δ, and TCR- and GGR-deficient and rad26Δ rad7Δ served as controls (empty symbols). Double mutants carrying the hpr1Δ and tho2Δ mutations in combination with the rad7Δ mutations were used as marker of phenotype strength. Data for the rad26Δ control strain were taken from (42) (dash line). Average values from three independent experiments are plotted for each genotype.
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Figure 1: mRNA export deficient cells are sensitive to UV in the absence of global genome repair. UV sensitivity curves of sub2-206, mex67-5, thp1Δ, sac3Δ, hpr1-101, and rad7Δ single and double mutants (plain symbols). Isogenic wild-type, NER-deficient rad1Δ, and TCR- and GGR-deficient and rad26Δ rad7Δ served as controls (empty symbols). Double mutants carrying the hpr1Δ and tho2Δ mutations in combination with the rad7Δ mutations were used as marker of phenotype strength. Data for the rad26Δ control strain were taken from (42) (dash line). Average values from three independent experiments are plotted for each genotype.

Mentions: To analyze whether defects in mRNP biogenesis and export result in impaired TCR, we studied mutants defective in both GGR and mRNA export. Isogenic sub2-206, mex67-5, rad7Δ, sub2-206 rad7Δ, mex67-5 rad7Δ mutants were generated and survival after UV irradiation was determined (Figure 1, upper panel). Isogenic repair-proficient W303-1A, repair-deficient rad1Δ and TCR-deficient rad26Δ rad7Δ strains were used as controls. The sub2-206 and mex65-7 single mutants show no increased UV sensitivity as compared to wild-type cells. However, upon UV irradiation viability of the sub2-206 rad7Δ and mex67-5 rad7Δ double mutants dropped below the levels of the rad7Δ single mutant. Survival of the sub2-206 rad7Δ strain was similar to survival of the rad26Δ rad7Δ strain, whereas mex67-5 rad7Δ was less affected. Next, we analyzed mutants of the Thp1-Sac3 complex, which acts downstream of Mex67-Mtr2 on the mRNP biogenesis and export route. Isogenic thp1Δ, sac3Δ, thp1Δ rad7Δ, and sac3Δ rad7Δ mutants were generated and UV survival was determined (Figure 1, middle panel). The thp1Δ and sac3Δ single mutants show no increased UV sensitivity as compared with wild-type cells. However, viability of the thp1Δ rad7Δ and sac3Δ rad7Δ double mutants was below the level of the rad7Δ single mutant upon UV irradiation.Figure 1.


A new connection of mRNP biogenesis and export with transcription-coupled repair.

Gaillard H, Wellinger RE, Aguilera A - Nucleic Acids Res. (2007)

mRNA export deficient cells are sensitive to UV in the absence of global genome repair. UV sensitivity curves of sub2-206, mex67-5, thp1Δ, sac3Δ, hpr1-101, and rad7Δ single and double mutants (plain symbols). Isogenic wild-type, NER-deficient rad1Δ, and TCR- and GGR-deficient and rad26Δ rad7Δ served as controls (empty symbols). Double mutants carrying the hpr1Δ and tho2Δ mutations in combination with the rad7Δ mutations were used as marker of phenotype strength. Data for the rad26Δ control strain were taken from (42) (dash line). Average values from three independent experiments are plotted for each genotype.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919492&req=5

Figure 1: mRNA export deficient cells are sensitive to UV in the absence of global genome repair. UV sensitivity curves of sub2-206, mex67-5, thp1Δ, sac3Δ, hpr1-101, and rad7Δ single and double mutants (plain symbols). Isogenic wild-type, NER-deficient rad1Δ, and TCR- and GGR-deficient and rad26Δ rad7Δ served as controls (empty symbols). Double mutants carrying the hpr1Δ and tho2Δ mutations in combination with the rad7Δ mutations were used as marker of phenotype strength. Data for the rad26Δ control strain were taken from (42) (dash line). Average values from three independent experiments are plotted for each genotype.
Mentions: To analyze whether defects in mRNP biogenesis and export result in impaired TCR, we studied mutants defective in both GGR and mRNA export. Isogenic sub2-206, mex67-5, rad7Δ, sub2-206 rad7Δ, mex67-5 rad7Δ mutants were generated and survival after UV irradiation was determined (Figure 1, upper panel). Isogenic repair-proficient W303-1A, repair-deficient rad1Δ and TCR-deficient rad26Δ rad7Δ strains were used as controls. The sub2-206 and mex65-7 single mutants show no increased UV sensitivity as compared to wild-type cells. However, upon UV irradiation viability of the sub2-206 rad7Δ and mex67-5 rad7Δ double mutants dropped below the levels of the rad7Δ single mutant. Survival of the sub2-206 rad7Δ strain was similar to survival of the rad26Δ rad7Δ strain, whereas mex67-5 rad7Δ was less affected. Next, we analyzed mutants of the Thp1-Sac3 complex, which acts downstream of Mex67-Mtr2 on the mRNP biogenesis and export route. Isogenic thp1Δ, sac3Δ, thp1Δ rad7Δ, and sac3Δ rad7Δ mutants were generated and UV survival was determined (Figure 1, middle panel). The thp1Δ and sac3Δ single mutants show no increased UV sensitivity as compared with wild-type cells. However, viability of the thp1Δ rad7Δ and sac3Δ rad7Δ double mutants was below the level of the rad7Δ single mutant upon UV irradiation.Figure 1.

Bottom Line: Careful analysis revealed that THO mutants are also specifically affected in TCR.Along with severe UV damage-dependent loss in processivity, RNAPII was found binding to chromatin upon UV irradiation in THO mutants, suggesting that RNAPII remains stalled at DNA lesions.Our results indicate that RNAPII is not proficient for TCR in mRNP biogenesis and export mutants, opening new perspectives on our knowledge of TCR in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Facultad de Biología, Universidad de Sevilla, CABIMER, CSIC-Universidad de Sevilla, Avenida Américo Vespucio s/n, 41092 Sevilla, Spain.

ABSTRACT
Although DNA repair is faster in the transcribed strand of active genes, little is known about the possible contribution of mRNP biogenesis and export in transcription-coupled repair (TCR). Interestingly, mutants of THO, a transcription complex involved in maintenance of genome integrity, mRNP biogenesis and export, were recently found to be deficient in nucleotide excision repair. In this study we show by molecular DNA repair analysis, that Sub2-Yra1 and Thp1-Sac3, two main mRNA export complexes, are required for efficient TCR in yeast. Careful analysis revealed that THO mutants are also specifically affected in TCR. Ribozyme-mediated mRNA self-cleavage between two hot spots for UV damage showed that efficient TCR does not depend on the nascent mRNA, neither in wild-type nor in mutant cells. Along with severe UV damage-dependent loss in processivity, RNAPII was found binding to chromatin upon UV irradiation in THO mutants, suggesting that RNAPII remains stalled at DNA lesions. Furthermore, Def1, a factor responsible for the degradation of stalled RNAPII, appears essential for the viability of THO mutants subjected to DNA damage. Our results indicate that RNAPII is not proficient for TCR in mRNP biogenesis and export mutants, opening new perspectives on our knowledge of TCR in eukaryotic cells.

Show MeSH
Related in: MedlinePlus