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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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Synergistic increase of temperature and UV sensitivity phenotypes in def1 hpr1 double mutants. (A) Growth of isogenic W303 yeast strains carrying single and double combinations of the hpr1 and def1 mutations at 30°C (left panel) and at 37°C (right panel). (B and C) UV sensitivity curves of isogenic hpr1Δ, def1Δ, and rad7Δ single, double and triple mutants. Isogenic wild-type and NER-deficient rad1Δ strain were used as controls. Note that the UV dose used in panel C was lower than in panel B. Description is as in Figure 1.
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Figure 6: Synergistic increase of temperature and UV sensitivity phenotypes in def1 hpr1 double mutants. (A) Growth of isogenic W303 yeast strains carrying single and double combinations of the hpr1 and def1 mutations at 30°C (left panel) and at 37°C (right panel). (B and C) UV sensitivity curves of isogenic hpr1Δ, def1Δ, and rad7Δ single, double and triple mutants. Isogenic wild-type and NER-deficient rad1Δ strain were used as controls. Note that the UV dose used in panel C was lower than in panel B. Description is as in Figure 1.

Mentions: Since we can rule out an active role of the nascent mRNA in TCR, it is conceivable that an intact RNAPII complex is sufficient to mediate proficient TCR. Recently, a protein called Def1 was shown to trigger ubiquitylation and degradation of RNAPII in response to UV damage as an alternative pathway to DNA repair (26,27,53). To study the possible requirement for Def1 in the removal of trapped RNAPII presumably present in THO mutants, hpr1Δ def1Δ double mutants were generated and analyzed. hpr1Δ def1Δ double mutants were viable, but very slow growing. Since all THO/TREX mutants grow poorly at 37°C, we first investigated whether deletion of DEF1 might increase the temperature sensitivity (ts) phenotype of hpr1Δ cells (Figure 6A). An additive effect that made cells inviable at 37°C was observed in the double mutant as compared to the single mutant. Next, we performed UV survival curves in isogenic def1Δ, hpr1Δ and def1Δ hpr1Δ mutants (Figure 6B). Viability of the def1Δ hpr1Δ double mutants was reduced below the levels of the def1Δ and hpr1Δ single mutants upon UV irradiation, indicating a synergistic effect of the two mutations on UV sensitivity. In the absence of GGR, def1Δ has been shown to be highly sensitive to UV irradiation (53). Nevertheless, the UV sensitivity of def1Δ hpr1Δ rad7Δ cells was increased as compared to def1Δ rad7Δ cells (Figure 6B and C). These genetic interactions between DEF1 and HPR1 indicate that Def1 is important for the viability of THO mutants subjected to stress and DNA damage.Figure 6.


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

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

Synergistic increase of temperature and UV sensitivity phenotypes in def1 hpr1 double mutants. (A) Growth of isogenic W303 yeast strains carrying single and double combinations of the hpr1 and def1 mutations at 30°C (left panel) and at 37°C (right panel). (B and C) UV sensitivity curves of isogenic hpr1Δ, def1Δ, and rad7Δ single, double and triple mutants. Isogenic wild-type and NER-deficient rad1Δ strain were used as controls. Note that the UV dose used in panel C was lower than in panel B. Description is as in Figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Synergistic increase of temperature and UV sensitivity phenotypes in def1 hpr1 double mutants. (A) Growth of isogenic W303 yeast strains carrying single and double combinations of the hpr1 and def1 mutations at 30°C (left panel) and at 37°C (right panel). (B and C) UV sensitivity curves of isogenic hpr1Δ, def1Δ, and rad7Δ single, double and triple mutants. Isogenic wild-type and NER-deficient rad1Δ strain were used as controls. Note that the UV dose used in panel C was lower than in panel B. Description is as in Figure 1.
Mentions: Since we can rule out an active role of the nascent mRNA in TCR, it is conceivable that an intact RNAPII complex is sufficient to mediate proficient TCR. Recently, a protein called Def1 was shown to trigger ubiquitylation and degradation of RNAPII in response to UV damage as an alternative pathway to DNA repair (26,27,53). To study the possible requirement for Def1 in the removal of trapped RNAPII presumably present in THO mutants, hpr1Δ def1Δ double mutants were generated and analyzed. hpr1Δ def1Δ double mutants were viable, but very slow growing. Since all THO/TREX mutants grow poorly at 37°C, we first investigated whether deletion of DEF1 might increase the temperature sensitivity (ts) phenotype of hpr1Δ cells (Figure 6A). An additive effect that made cells inviable at 37°C was observed in the double mutant as compared to the single mutant. Next, we performed UV survival curves in isogenic def1Δ, hpr1Δ and def1Δ hpr1Δ mutants (Figure 6B). Viability of the def1Δ hpr1Δ double mutants was reduced below the levels of the def1Δ and hpr1Δ single mutants upon UV irradiation, indicating a synergistic effect of the two mutations on UV sensitivity. In the absence of GGR, def1Δ has been shown to be highly sensitive to UV irradiation (53). Nevertheless, the UV sensitivity of def1Δ hpr1Δ rad7Δ cells was increased as compared to def1Δ rad7Δ cells (Figure 6B and C). These genetic interactions between DEF1 and HPR1 indicate that Def1 is important for the viability of THO mutants subjected to stress and DNA damage.Figure 6.

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