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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03221

Journal of Cell Science 119, 5178-5192 (2006)
Published by The Company of Biologists 2006
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Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length

Robert N. Woolstencroft1,2, Traude H. Beilharz3, Michael A. Cook1,2, Thomas Preiss3,4, Daniel Durocher1,2,* and Mike Tyers1,2,*

1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, Canada
2 Graduate Department of Molecular and Medical Genetics, University of Toronto, Toronto, M5S 1A8, Canada
3 Victor Chang Cardiac Research Institute (VCCRI), Darlinghurst (Sydney), NSW 2010, Australia
4 School of Biotechnology and Biomolecular Sciences and St Vincent's Clinical School, University of New South Wales, Sydney, NSW 2052, Australia


Figure 1
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  		Fig. 1. Multiple genome-wide screens identify common genes required for tolerance to replication stress. (A) Gene deletion strains hypersensitive to hydroxyurea (HU) identified by the approaches of Bennett et al., Hartman and Tippery, Parsons et al. and this study were compiled to identify the shared HU sensitive strains (Bennett et al., 2001Go; Hartman and Tippery, 2004Go; Parsons et al., 2004Go). Number of gene deletions shared between datasets (intersections) or unique to each individual dataset (peripheries) are indicated. (B) HU-sensitive gene deletion mutants identified by at least three of the four approaches were classified by Gene Ontology (GO) process according to the Biological General Repository for Interaction Datasets (BioGRID; www.thebiogrid.org) (Stark et al., 2006Go). The network was created with Osprey visualization software (Breitkreutz et al., 2003Go).

 

Figure 2
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  		Fig. 2. Ccr4 is required for tolerance to replication stress and acts independently of the Dun1 checkpoint kinase. (A) HU sensitivity of a ccr4{Delta} (MT3768) strain transformed with a pRS416 (CEN) plasmid bearing wild-type CCR4; single mutations that abolish in vitro deadenylase activity (ccr4[E556A]; ccr4[D713A]); or combined double mutations (ccr4[E556A, D713A]) were assessed by growth of serial dilutions spotted onto media with or without 100 mM HU for 3 days. (B) Sensitivity of ccr4{Delta} (MT3768), dun1{Delta} (MT3769) or ccr4{Delta} dun1{Delta} (MT3772) strains was analyzed by serial dilution on indicated concentrations of HU as above. (C) Irreversible HU sensitivity was assayed by incubating the indicated strains in liquid media containing 200 mM HU. At indicated time-points, aliquots were removed and cells plated onto rich media lacking HU using a spiral plating system. Colony-forming units (CFU) were determined after incubation for 3 days.

 

Figure 3
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  		Fig. 3. Rad53 activity persists after HU exposure in cells lacking both CCR4 and DUN1. (A) Wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769) and ccr4{Delta} dun1{Delta} (MT3772) strains were treated with 200 mM HU for 3 hours followed by assessment of Rad53 kinase activity by in situ assay (ISA), as manifest in Rad53 autophosphorylation in vitro (upper panel), and by the extent of phosphorylation-dependent Rad53 mobility shift, as detected by the DAB001 Rad53 antibody (lower panel). (B) Cell cycle progression after replication stress is severely delayed in a ccr4{Delta} dun1{Delta} strain. DNA content of the strains in A was determined by FACS analysis of either asynchronous mid-log phase cultures (ASN) or after S-phase synchronization in 200 mM HU for 3 hours (+HU) followed by washout of HU for the indicated times.

 

Figure 4
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  		Fig. 4. Genetic interactions between CCR4, the replication checkpoint and the DNA replication machinery. (A) Deletion of CCR4 is synthetic lethal with loss of RNR1 or RNR4. The diploid strains MT3802 (MATa/{alpha} ccr4{Delta} rnr4{Delta}) and MT3809 (MATa/{alpha} ccr4{Delta} rnr1{Delta}) were sporulated, dissected and genotyped (27 tetrads for ccr4{Delta}/rnr1{Delta} cross and 36 tetrads for ccr4{Delta}/rnr4{Delta} cross). Synthetic lethal interactions (ccr4{Delta} rnr1{Delta} or ccr4{Delta} rnr4{Delta}) were inferred from antibiotic resistance marker segregation (arrows). At least one isolate for each possible genotype is depicted. (B) ccr4{Delta} exacerbates the phenotypes of checkpoint and replication mutants. HU sensitivity of the indicated mutants was assessed by serial dilution on media containing indicated concentrations of HU, followed by growth for 3 days. Growth of pri1-M4 and ccr4{Delta} pri1-M4 (MT3799) strains was assessed on rich media, followed by incubation for 3 days at the indicated temperatures. (C) CCR4 acts independently of Rad53 and Mec1, but may act in concert with CHK1. The indicated checkpoint kinase mutations and strains were analyzed for HU sensitivity as above. (D) Quantitative assessment of sensitivity of the indicated strains to the indicated concentrations of HU was determined with a spiral plating system and CFU determined after 3-5 days growth.

 

Figure 5
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  		Fig. 5. Deletion of CRT1 or PBP1 suppresses the HU sensitivity of a ccr4{Delta} strain. (A) ccr4{Delta} crt1{Delta} (MT3771), ccr4{Delta} pbp1{Delta} (MT3777), wild-type (BY4741), ccr4{Delta} (MT3768), crt1{Delta} (MT3770) and pbp1{Delta} (MT3776) strains were tested for HU sensitivity by serial dilution analysis, as described in Fig. 2. (B) Indicated strains were assessed for irreversible HU sensitivity using a spiral plating system, as described in Fig. 2C. (C) Rad53 activity and phosphorylation were assessed in ccr4{Delta} dun1{Delta} (MT3772), ccr4{Delta} dun1{Delta} crt1{Delta} (RSYS07) and wild-type (BY4741) strains after exposure to 0.2 M HU for 3 hours, as described for Fig. 3.

 

Figure 6
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  		Fig. 6. Ccr4 regulates CRT1 mRNA poly(A) tail length. (A) Poly(A) tail lengths were determined by LM-PAT analysis of total cellular RNA isolated from isogenic ccr4{Delta} (MT3768) and wild-type (BY4741), and from isogenic ccr4-1 (Y359) and wild-type (Y136) strain pairs, each either untreated or treated with 100 mM HU for 90 minutes. The estimated number of adenosine residues in the poly(A) tails from ccr4{Delta} strains are noted for specific transcripts. Notice that a segment of 12 adenosines is introduced by the linker primer into all cDNA produced, and thus this length represents the lowest limit of PCR product size from short tailed mRNAs. (B) CRT1 mRNA levels were determined by quantitative Real Time PCR of total RNA isolated from wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769) or ccr4{Delta} dun1{Delta} (MT3772) strains that were either untreated or treated with 100 mM HU for 90 minutes. CRT1 mRNA levels were normalized to ACT1 mRNA levels and reported as the fraction of untreated wild-type CRT1 mRNA abundance. (C) Alteration of CRT1 mRNA poly(A) tail length in wild-type (Y136), ccr4-1 (Y359), ccr4{Delta} (Y294), caf1{Delta} (Y297), pan2{Delta} (YTP1), ccr4-1 pan2{Delta} (YTP2), ccr4{Delta} (MT3768) and wild-type (BY4741) strains by LM-PAT analysis of total RNA. (D) Crt1 protein abundance was determined in wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769) and ccr4{Delta} dun1{Delta} (MT3772) strains that were either untreated or treated with 100 mM HU for 90 minutes. Crt1 was detected with polyclonal anti-Crt1 antibody. Strains crt1{Delta} (MT3770) and ccr4{Delta} dun1{Delta} crt1{Delta} (MT3773) served as negative controls for antibody specificity; equivalent protein loading was assessed with a monoclonal anti-Pgk1 antibody. (E) CRT1 mRNA poly(A) tail lengths were determined in wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769), ccr4{Delta} dun1{Delta} (MT3772), pbp1{Delta} (MT3776), ccr4{Delta} pbp1{Delta} (MT3777), dun1{Delta} pbp1{Delta} (MT3842), ccr4{Delta} dun1{Delta} pbp1{Delta} (MT3843), chk1{Delta} (MT3784) and ccr4{Delta} chk1{Delta} (MT3785) strains by LM-PAT analysis of total RNA. The number of adenosine residues for major poly(A) tail species for specific transcripts are indicated. (F) The effect of CRT1 overexpression on tolerance to replication stress was determined with an integrated GAL-CRT1 allele in strains GAL1-CRT1 (MT3806), ccr4{Delta} GAL1-CRT1 (MT3807) and dun1{Delta} GAL1-CRT1 (MT3808). Sensitivity to HU was determined by serial dilution and spotting on indicated concentrations of HU under conditions of GAL1-CRT1 repression (+GLU) or induction (+GAL). Wild-type (BY4741), ccr4{Delta} (MT3768), dun1{Delta} (MT3769) and ccr4{Delta} dun1{Delta} (MT3772) strains were used as controls.

 

Figure 7
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  		Fig. 7. Crt1 controls transcription of a suite of genes that may affect viability during replication stress. (A) Genome-wide transcriptional profiles were obtained by competitive hybridization of labeled cDNAs from ccr4{Delta} crt1{Delta} (MT3771, Cy5) vs. ccr4{Delta} (MT3768, Cy3) and for ccr4{Delta} dun1{Delta} crt1{Delta} (MT3773, Cy5) vs. ccr4{Delta} dun1{Delta} (MT3772, Cy3), as described in Materials and Methods. See Table 2 for specific gene induction/repression values. (B) Top differentially regulated genes between indicated strains, with and without HU treatment. Scale shows relative induction (red) or repression (green) with respect to control samples. (C) Effect of overexpression of RNR genes on sensitivity of the ccr4{Delta} dun1{Delta} double mutant to HU. (Top panels) Overexpression of RNR3. Strain ccr4{Delta} dun1{Delta} trp1{Delta} (MT3800) was transformed with either pGAP (pBAD054 TRP1, empty) or pGAP-RNR3 (pBAD079 TRP1, RNR3). ccr4{Delta} dun1{Delta} (MT3772), ccr4{Delta} dun1{Delta} crt1{Delta} (MT3773) and wild-type (BY4741) strains are shown for comparison. Strains were serially diluted, spotted on media lacking tryptophan (-trp) and containing indicated concentrations of HU and grown for 3-5 days. (Middle and bottom panels) Coordinate overexpression of multiple RNR subunits. Strain ccr4{Delta} dun1{Delta} trp1{Delta} (MT3800) was transformed with either pGAP (pBAD054 TRP1, empty vector) or pGAP-RNR3 (pBAD079 TRP1, RNR3) in combination with pGAL-RNR4 (pDL57 URA3, RNR4) and either pGAL-FLAG (pMT3164 LEU2, empty vector) or pGAL-RNR2-FLAG (pMT3963 LEU2, RNR2-FLAG). ccr4{Delta} dun1{Delta} crt1{Delta} (MT3773) was co-transformed with pGAL (pDL54 URA3, empty vector) or pGAL-FLAG (pMT3164 LEU2, empty vector) as a comparison. Strains were serially diluted, spotted on media lacking histidine, leucine, uracil and tryptophan (-HLUW) and containing indicated concentrations of HU and either glucose (GLU) or galactose (GAL) and grown for 3-5 days.

 

Figure 8
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  		Fig. 8. Ccr4 and Dun1 collaborate to inhibit Crt1 activity and to promote induction of the DNA-damage gene regulon after replication stress. Ccr4 regulates CRT1 mRNA poly(A) tail length, which may influence translation and thereby levels of Crt1 protein. The Mec1-Rad53-Dun1 checkpoint kinase cascade phosphorylates and inhibits Crt1 repressor activity at DNA-damage-inducible promoters. Loss of either regulatory branch results in HU sensitivity; simultaneous loss of both regulatory branches causes severe and irreversible HU lethality.

 

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© The Company of Biologists Ltd 2006