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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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Cleavage of the nascent mRNA does not restore TCR in cells defective in THO. (A) Southern blot analysis of strand-specific repair of a 2.2-kb LacZ fragment containing either the active (Rib+) or mutated Rib (ribm) between two T-tracts in tho2Δ cells after UV irradiation (230 J/m2). The initial damage averaged 0.5 ± 0.07 CPD/Kb. In the T-tracts, the initial damage corresponded to 1.05% (T1, Rib+), 2.51% (T1, Rib+), 1.55% (T1, ribm) and 2.2% (T2, ribm) of the respective total lane signal. Description is as in Figure 4C. (B) Graphical representation of the repair analysis as shown in A and in Figure 4D. Repair of the intact restriction fragment in wild-type and tho2 cells (left panel) and of the T-tracts in tho2 cells (right panel) are plotted. The percentage of repair was determined from the signal intensities as described in ‘Materials and Methods’. Average values derived from two independent experiments are plotted.
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Figure 5: Cleavage of the nascent mRNA does not restore TCR in cells defective in THO. (A) Southern blot analysis of strand-specific repair of a 2.2-kb LacZ fragment containing either the active (Rib+) or mutated Rib (ribm) between two T-tracts in tho2Δ cells after UV irradiation (230 J/m2). The initial damage averaged 0.5 ± 0.07 CPD/Kb. In the T-tracts, the initial damage corresponded to 1.05% (T1, Rib+), 2.51% (T1, Rib+), 1.55% (T1, ribm) and 2.2% (T2, ribm) of the respective total lane signal. Description is as in Figure 4C. (B) Graphical representation of the repair analysis as shown in A and in Figure 4D. Repair of the intact restriction fragment in wild-type and tho2 cells (left panel) and of the T-tracts in tho2 cells (right panel) are plotted. The percentage of repair was determined from the signal intensities as described in ‘Materials and Methods’. Average values derived from two independent experiments are plotted.

Mentions: Nevertheless, in contrast to wild-type cells, the occurrence of sub-optimal mRNP might impede the process of TCR in mutants of THO/TREX and Thp1-Sac3. To test this possibility, we used the ribozyme system to assess whether wild-type TCR can be restored in T2 after ribozyme self-cleavage of the nascent mRNA in tho2Δ and thp1Δ cells (Figure 5). Repair in the full length LacZ fragment was clearly below repair levels achieved in wild-type cells, confirming the TCR deficiency of tho2Δ cells. Comparison of repair efficiencies in T1 and T2 did not show any significant difference, neither in the active (Rib+) nor in the mutated (ribm) ribozyme constructs. Similar results were obtained in thp1Δ cells (data not shown). Thus, our results indicate that cleavage of the nascent mRNA does not restore TCR in THO and Thp1 mutants. Consequently, we assume that the key player in the organization of TCR is the RNAPII itself, or some associated factors, rather than the nascent mRNA.Figure 5.


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

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

Cleavage of the nascent mRNA does not restore TCR in cells defective in THO. (A) Southern blot analysis of strand-specific repair of a 2.2-kb LacZ fragment containing either the active (Rib+) or mutated Rib (ribm) between two T-tracts in tho2Δ cells after UV irradiation (230 J/m2). The initial damage averaged 0.5 ± 0.07 CPD/Kb. In the T-tracts, the initial damage corresponded to 1.05% (T1, Rib+), 2.51% (T1, Rib+), 1.55% (T1, ribm) and 2.2% (T2, ribm) of the respective total lane signal. Description is as in Figure 4C. (B) Graphical representation of the repair analysis as shown in A and in Figure 4D. Repair of the intact restriction fragment in wild-type and tho2 cells (left panel) and of the T-tracts in tho2 cells (right panel) are plotted. The percentage of repair was determined from the signal intensities as described in ‘Materials and Methods’. Average values derived from two independent experiments are plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Cleavage of the nascent mRNA does not restore TCR in cells defective in THO. (A) Southern blot analysis of strand-specific repair of a 2.2-kb LacZ fragment containing either the active (Rib+) or mutated Rib (ribm) between two T-tracts in tho2Δ cells after UV irradiation (230 J/m2). The initial damage averaged 0.5 ± 0.07 CPD/Kb. In the T-tracts, the initial damage corresponded to 1.05% (T1, Rib+), 2.51% (T1, Rib+), 1.55% (T1, ribm) and 2.2% (T2, ribm) of the respective total lane signal. Description is as in Figure 4C. (B) Graphical representation of the repair analysis as shown in A and in Figure 4D. Repair of the intact restriction fragment in wild-type and tho2 cells (left panel) and of the T-tracts in tho2 cells (right panel) are plotted. The percentage of repair was determined from the signal intensities as described in ‘Materials and Methods’. Average values derived from two independent experiments are plotted.
Mentions: Nevertheless, in contrast to wild-type cells, the occurrence of sub-optimal mRNP might impede the process of TCR in mutants of THO/TREX and Thp1-Sac3. To test this possibility, we used the ribozyme system to assess whether wild-type TCR can be restored in T2 after ribozyme self-cleavage of the nascent mRNA in tho2Δ and thp1Δ cells (Figure 5). Repair in the full length LacZ fragment was clearly below repair levels achieved in wild-type cells, confirming the TCR deficiency of tho2Δ cells. Comparison of repair efficiencies in T1 and T2 did not show any significant difference, neither in the active (Rib+) nor in the mutated (ribm) ribozyme constructs. Similar results were obtained in thp1Δ cells (data not shown). Thus, our results indicate that cleavage of the nascent mRNA does not restore TCR in THO and Thp1 mutants. Consequently, we assume that the key player in the organization of TCR is the RNAPII itself, or some associated factors, rather than the nascent mRNA.Figure 5.

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