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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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An intact nascent mRNA is not required for TCR in wild-type yeast cells. (A) Expression of a modified LacZ containing the self-cleaving Hammerhead ribozyme (Rib) between two 39-bp long T-tracts was placed under the control of the GAL1 promoter. The first (T1) and the second (T2) T-tracts as well as the Rib sequence are indicated. (B) Northern analysis of LacZ mRNA isolated from plasmids containing either an active (Rib+) or a mutated form of Rib (ribm). The apparent difference in signal intensity between the two constructs probably results from distinct transcript stability, unequal transfer efficiency of short and long RNA, as well as different hybridization efficiency due to the long transcript migrating very close to the abundant rRNA. (C) Southern blot analysis of representative experiments showing repair of a 2.2 kb (HpaI/EcoRI) LacZ fragment containing either the active Rib+ (LacZ-Rib+) or the mutated ribm (LacZ-ribm) in W303 cells after UV irradiation (230 J/m2). Description is as in Figure 2A, except that genomic DNA was probed for the TS of LacZ. The initial damage averaged 0.3 ± 0.005 CPD/Kb. In the T-tracts, the initial damage corresponded to 0.54% (T1, Rib+), 1.08% (T2, Rib+), 0.79% (T1, ribm) and 1.03% (T2, ribm) of the respective total lane signal. (D) Graphical representation of the T-tracts repair analysis as shown in C. 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 4: An intact nascent mRNA is not required for TCR in wild-type yeast cells. (A) Expression of a modified LacZ containing the self-cleaving Hammerhead ribozyme (Rib) between two 39-bp long T-tracts was placed under the control of the GAL1 promoter. The first (T1) and the second (T2) T-tracts as well as the Rib sequence are indicated. (B) Northern analysis of LacZ mRNA isolated from plasmids containing either an active (Rib+) or a mutated form of Rib (ribm). The apparent difference in signal intensity between the two constructs probably results from distinct transcript stability, unequal transfer efficiency of short and long RNA, as well as different hybridization efficiency due to the long transcript migrating very close to the abundant rRNA. (C) Southern blot analysis of representative experiments showing repair of a 2.2 kb (HpaI/EcoRI) LacZ fragment containing either the active Rib+ (LacZ-Rib+) or the mutated ribm (LacZ-ribm) in W303 cells after UV irradiation (230 J/m2). Description is as in Figure 2A, except that genomic DNA was probed for the TS of LacZ. The initial damage averaged 0.3 ± 0.005 CPD/Kb. In the T-tracts, the initial damage corresponded to 0.54% (T1, Rib+), 1.08% (T2, Rib+), 0.79% (T1, ribm) and 1.03% (T2, ribm) of the respective total lane signal. (D) Graphical representation of the T-tracts repair analysis as shown in C. 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: The molecular basis underlying the requirement of functional mRNP biogenesis and export factors for TCR might rely on the proper packaging of the nascent transcript or on their effect on transcription. In repair-proficient cells, the nascent mRNA could mediate the TCR reaction in response to the transcriptional stalling occurring at DNA lesions. To test this possibility, we designed a construct containing two 39-bp long T-tract sequences inserted at different sites within the GAL1 promoter-driven LacZ ORF. Between the two T-tract sequences, 52 bp encoding either an active self-cleaving Hammerhead ribozyme or an inactive mutated form (47) were inserted (Figure 4A). Northern analysis confirmed a complete disappearance of the full length mRNA in the construct carrying the active ribozyme (Figure 4B), indicating that the nascent mRNA was efficiently cleaved between the T-tracts.Figure 4.


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

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

An intact nascent mRNA is not required for TCR in wild-type yeast cells. (A) Expression of a modified LacZ containing the self-cleaving Hammerhead ribozyme (Rib) between two 39-bp long T-tracts was placed under the control of the GAL1 promoter. The first (T1) and the second (T2) T-tracts as well as the Rib sequence are indicated. (B) Northern analysis of LacZ mRNA isolated from plasmids containing either an active (Rib+) or a mutated form of Rib (ribm). The apparent difference in signal intensity between the two constructs probably results from distinct transcript stability, unequal transfer efficiency of short and long RNA, as well as different hybridization efficiency due to the long transcript migrating very close to the abundant rRNA. (C) Southern blot analysis of representative experiments showing repair of a 2.2 kb (HpaI/EcoRI) LacZ fragment containing either the active Rib+ (LacZ-Rib+) or the mutated ribm (LacZ-ribm) in W303 cells after UV irradiation (230 J/m2). Description is as in Figure 2A, except that genomic DNA was probed for the TS of LacZ. The initial damage averaged 0.3 ± 0.005 CPD/Kb. In the T-tracts, the initial damage corresponded to 0.54% (T1, Rib+), 1.08% (T2, Rib+), 0.79% (T1, ribm) and 1.03% (T2, ribm) of the respective total lane signal. (D) Graphical representation of the T-tracts repair analysis as shown in C. 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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Related In: Results  -  Collection

License
Show All Figures
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Figure 4: An intact nascent mRNA is not required for TCR in wild-type yeast cells. (A) Expression of a modified LacZ containing the self-cleaving Hammerhead ribozyme (Rib) between two 39-bp long T-tracts was placed under the control of the GAL1 promoter. The first (T1) and the second (T2) T-tracts as well as the Rib sequence are indicated. (B) Northern analysis of LacZ mRNA isolated from plasmids containing either an active (Rib+) or a mutated form of Rib (ribm). The apparent difference in signal intensity between the two constructs probably results from distinct transcript stability, unequal transfer efficiency of short and long RNA, as well as different hybridization efficiency due to the long transcript migrating very close to the abundant rRNA. (C) Southern blot analysis of representative experiments showing repair of a 2.2 kb (HpaI/EcoRI) LacZ fragment containing either the active Rib+ (LacZ-Rib+) or the mutated ribm (LacZ-ribm) in W303 cells after UV irradiation (230 J/m2). Description is as in Figure 2A, except that genomic DNA was probed for the TS of LacZ. The initial damage averaged 0.3 ± 0.005 CPD/Kb. In the T-tracts, the initial damage corresponded to 0.54% (T1, Rib+), 1.08% (T2, Rib+), 0.79% (T1, ribm) and 1.03% (T2, ribm) of the respective total lane signal. (D) Graphical representation of the T-tracts repair analysis as shown in C. 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: The molecular basis underlying the requirement of functional mRNP biogenesis and export factors for TCR might rely on the proper packaging of the nascent transcript or on their effect on transcription. In repair-proficient cells, the nascent mRNA could mediate the TCR reaction in response to the transcriptional stalling occurring at DNA lesions. To test this possibility, we designed a construct containing two 39-bp long T-tract sequences inserted at different sites within the GAL1 promoter-driven LacZ ORF. Between the two T-tract sequences, 52 bp encoding either an active self-cleaving Hammerhead ribozyme or an inactive mutated form (47) were inserted (Figure 4A). Northern analysis confirmed a complete disappearance of the full length mRNA in the construct carrying the active ribozyme (Figure 4B), indicating that the nascent mRNA was efficiently cleaved between the T-tracts.Figure 4.

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