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Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1-Suc22 assembly.

Takahashi S, Kontani K, Araki Y, Katada T - Nucleic Acids Res. (2007)

Bottom Line: Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation.DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly.Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Appropriate supply of deoxyribonucleotides by the ribonucleotide reductase (RNR) complex is essential for DNA replication and repair. One recent model for the RNR activation in Schizosaccharomyces pombe is translocation of the regulatory subunit Suc22 from the nucleoplasm to the cytoplasm. The RNR inhibitory protein Spd1, which retains Suc22 in the nucleoplasm, is rapidly degraded upon DNA-replication stress, resulting in release of Suc22 to form the active RNR complex in the cytoplasm. Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation. Caf1 is required not only for the stress-induced translocation of Suc22 from nucleoplasm to cytoplasm but also for the degradation of nucleoplasmic Spd1. DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly. Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

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Neither increase of 1.9-kb suc22 mRNA level nor deadenylation of mRNA poly(A) tails by Caf1 contributes to the HU-induced stress-response pathway. (A) Logarithmically growing cells, wild type (YSP001), not4Δ (YSP131), ccr4Δ (YSP002), caf1Δ (YSP066), caf1Δ pRep1 (YSP188), caf1Δ pRep1-Caf1 (YSP189) and caf1Δ pRep1-Caf1/D50A (YSP190), were incubated in the presence (+) and absence (−) of 10 mM HU for 2 h at 30°C. RNA preparation and northern blotting analysis were performed as described in the Materials and Methods section. Membranes were exposed to phosphorImager screens and followed by the quantitative analysis of suc22 mRNAs using Molecular Dynamics software. (B) Total RNA (1 μg) were purified from the above cells (YSP027, YSP188, YSP189 and YSP190) and end-labeled with 5′-[32P] pCp using T4 RNA ligase. The radiolabeled mRNAs were digested with RNase A and separated with 12% polyacrylamide–7.5 M urea gel electrophoresis.
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Figure 3: Neither increase of 1.9-kb suc22 mRNA level nor deadenylation of mRNA poly(A) tails by Caf1 contributes to the HU-induced stress-response pathway. (A) Logarithmically growing cells, wild type (YSP001), not4Δ (YSP131), ccr4Δ (YSP002), caf1Δ (YSP066), caf1Δ pRep1 (YSP188), caf1Δ pRep1-Caf1 (YSP189) and caf1Δ pRep1-Caf1/D50A (YSP190), were incubated in the presence (+) and absence (−) of 10 mM HU for 2 h at 30°C. RNA preparation and northern blotting analysis were performed as described in the Materials and Methods section. Membranes were exposed to phosphorImager screens and followed by the quantitative analysis of suc22 mRNAs using Molecular Dynamics software. (B) Total RNA (1 μg) were purified from the above cells (YSP027, YSP188, YSP189 and YSP190) and end-labeled with 5′-[32P] pCp using T4 RNA ligase. The radiolabeled mRNAs were digested with RNase A and separated with 12% polyacrylamide–7.5 M urea gel electrophoresis.

Mentions: In S. cerevisiae, mutations of Ccr4–Not complex impair the transcription of mRNAs coding RNR genes in response to HU (13). In accordance to previous work (41), HU induced the 1.9-kb suc22 mRNA in S. pombe wild-type cells (Figure 3A, lane 2). The increase of suc22 mRNA level was still observed in not4Δ (lane 4) and ccr4Δ (lane 6) cells, but it was totally abolished in caf1Δ cells (lane 8). Moreover, the defect of suc22 mRNA increase in caf1Δ cells was compensated not only by the introduction of wild-type Caf1 (lane 12) but also by the Caf1/D50A mutant (lane 14). It thus appeared that Caf1/D50A mutation is still capable of inducing the suc22 mRNA and that the ability of Caf1 to induce the 1.9-kb suc22 mRNA is not responsible for the HU-high sensitivity observed in caf1Δ and ccr4Δ cells.Figure 3.


Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1-Suc22 assembly.

Takahashi S, Kontani K, Araki Y, Katada T - Nucleic Acids Res. (2007)

Neither increase of 1.9-kb suc22 mRNA level nor deadenylation of mRNA poly(A) tails by Caf1 contributes to the HU-induced stress-response pathway. (A) Logarithmically growing cells, wild type (YSP001), not4Δ (YSP131), ccr4Δ (YSP002), caf1Δ (YSP066), caf1Δ pRep1 (YSP188), caf1Δ pRep1-Caf1 (YSP189) and caf1Δ pRep1-Caf1/D50A (YSP190), were incubated in the presence (+) and absence (−) of 10 mM HU for 2 h at 30°C. RNA preparation and northern blotting analysis were performed as described in the Materials and Methods section. Membranes were exposed to phosphorImager screens and followed by the quantitative analysis of suc22 mRNAs using Molecular Dynamics software. (B) Total RNA (1 μg) were purified from the above cells (YSP027, YSP188, YSP189 and YSP190) and end-labeled with 5′-[32P] pCp using T4 RNA ligase. The radiolabeled mRNAs were digested with RNase A and separated with 12% polyacrylamide–7.5 M urea gel electrophoresis.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851654&req=5

Figure 3: Neither increase of 1.9-kb suc22 mRNA level nor deadenylation of mRNA poly(A) tails by Caf1 contributes to the HU-induced stress-response pathway. (A) Logarithmically growing cells, wild type (YSP001), not4Δ (YSP131), ccr4Δ (YSP002), caf1Δ (YSP066), caf1Δ pRep1 (YSP188), caf1Δ pRep1-Caf1 (YSP189) and caf1Δ pRep1-Caf1/D50A (YSP190), were incubated in the presence (+) and absence (−) of 10 mM HU for 2 h at 30°C. RNA preparation and northern blotting analysis were performed as described in the Materials and Methods section. Membranes were exposed to phosphorImager screens and followed by the quantitative analysis of suc22 mRNAs using Molecular Dynamics software. (B) Total RNA (1 μg) were purified from the above cells (YSP027, YSP188, YSP189 and YSP190) and end-labeled with 5′-[32P] pCp using T4 RNA ligase. The radiolabeled mRNAs were digested with RNase A and separated with 12% polyacrylamide–7.5 M urea gel electrophoresis.
Mentions: In S. cerevisiae, mutations of Ccr4–Not complex impair the transcription of mRNAs coding RNR genes in response to HU (13). In accordance to previous work (41), HU induced the 1.9-kb suc22 mRNA in S. pombe wild-type cells (Figure 3A, lane 2). The increase of suc22 mRNA level was still observed in not4Δ (lane 4) and ccr4Δ (lane 6) cells, but it was totally abolished in caf1Δ cells (lane 8). Moreover, the defect of suc22 mRNA increase in caf1Δ cells was compensated not only by the introduction of wild-type Caf1 (lane 12) but also by the Caf1/D50A mutant (lane 14). It thus appeared that Caf1/D50A mutation is still capable of inducing the suc22 mRNA and that the ability of Caf1 to induce the 1.9-kb suc22 mRNA is not responsible for the HU-high sensitivity observed in caf1Δ and ccr4Δ cells.Figure 3.

Bottom Line: Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation.DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly.Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Appropriate supply of deoxyribonucleotides by the ribonucleotide reductase (RNR) complex is essential for DNA replication and repair. One recent model for the RNR activation in Schizosaccharomyces pombe is translocation of the regulatory subunit Suc22 from the nucleoplasm to the cytoplasm. The RNR inhibitory protein Spd1, which retains Suc22 in the nucleoplasm, is rapidly degraded upon DNA-replication stress, resulting in release of Suc22 to form the active RNR complex in the cytoplasm. Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation. Caf1 is required not only for the stress-induced translocation of Suc22 from nucleoplasm to cytoplasm but also for the degradation of nucleoplasmic Spd1. DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly. Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

Show MeSH
Related in: MedlinePlus