Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Acting through a complex signalling network, DNA lesions trigger a range of cellular responses including DNA repair, cell cycle arrest, altered gene expression and cell death, which help to limit the mutagenic effects of such DNA damage. RNA processing factors are increasingly being recognised as important targets of DNA damage signalling, with roles in the regulation of gene expression and also more directly in the promotion of DNA repair. In this study, we have used a Xenopus laevis egg extract system to analyse the DNA damage-dependent phosphorylation of a putative RNA export factor, Cip29. We have found that Cip29 is rapidly phosphorylated in response to DNA double-strand breaks in this experimental system. We show that the DNA damage-inducible modification of Cip29 is dependent on the activity of the key double-strand break response kinase, ATM, and we have identified a conserved serine residue as a damage-dependent phosphorylation site. Finally, we have determined that Cip29 is not required for efficient DNA end-joining in egg extracts. Taken together, these data identify Cip29 as a novel target of the DNA damage response and suggest that the damage-dependent modification of Cip29 may relate to a role in the regulation of gene expression after DNA damage.
???displayArticle.pubmedLink???
28715428
???displayArticle.pmcLink???PMC5513483 ???displayArticle.link???PLoS One
Fig 1. Detection of Cip29 and phosphorylated Cip29 proteins in X. laevis egg extract.(A) Immunodetection of Cip29 on a Western blot of X. laevis egg extract (E), in vitro translated Cip29 (T1) and in vitro translated Cip29b (T2). A non-specific band detected by the α-Cip29 antibodies in X. laevis egg extract is indicated with an asterisk. Molecular weight markers (kDa) are indicated on the left. (B) Western blot of Cip29 in X. laevis egg extract, undepleted or immunodepleted with either non-specific rabbit IgGs (mock) or α-Cip29 antibodies (left panel), and in the immunoprecipitated samples (right panel). The non-specific band (*) serves as a loading control for extract samples. (C) Amino acid sequence alignment of X. laevis Cip29 and Cip29b. (D) Western blots of egg extract incubated with or without 50ng/μl AT70 (21°C, 1h). Samples were resolved by electrophoresis on a standard 12% SDS-PAGE gel (left panel) and an 8% SDS-PAGE gel containing 15μM Phos-tag (right panel) before immunoblotting with the indicated antibodies. Modified Cip29 protein is denoted by Cip29-P (E) Cip29 was immunoprecipitated from egg extract supplemented with 50ng/μl AT70 (21°C, 1h). The immunoprecipitation reactions were treated with either 1 x NEBuffer 3 (-CIP), or NEBuffer 3 containing 10 units of CIP, or 10 units of heat-inactivated CIP (21°C, 1h) before glycine elution of the immunoprecipitated proteins. Eluted protein was supplemented with 2μg of Cip29-depleted extract before separation on Phos-tag SDS-PAGE, to optimise resolution of the modified form of Cip29.
Fig 2. Cip29 is phosphorylated in a DNA damage- and ATM-dependent manner in X. laevis egg extract.(A) Egg extract was supplemented with 50ng/μl AT70 and incubated at 21°C for the indicated times before reactions were stopped by addition of 5 x SDS-PAGE gel loading buffer. Samples were analysed by Phos-tag SDS-PAGE and western blotting with the indicated antibodies. (B) Egg extract was incubated with either AT70, cut plasmid or uncut plasmid (50ng/μl), in the absence or presence of caffeine (5mM) (21°C, 1h) before analysis by Phos-tag SDS-PAGE and western blotting. Dotted line indicates removal of intervening lanes on the same gel for the sake of clarity. (C) Egg extract was incubated in the absence or presence of 50ng/μl AT70 supplemented with 5mm caffeine, 10μM DNA-PKi (NU7441), 20μM and 40μM ATMi (KU55933) or 20μM and 40μM ATRi (VE821) as indicated (21°C, 1h), and samples were analysed by Phos-tag SDS-PAGE and western blotting. (D) Egg extract, immunodepleted with non-specific rabbit IgGs (mock), α-Chk1 or α-Chk2 antibodies, was incubated with 50ng/μl AT70 (21°C, 30min) and analysed by Phos-tag SDS-PAGE and western blotting. (E) Western blot of immunodepleted extracts from (D) indicating depletion efficiency for Chk1 and Chk2. Molecular weight markers (kDa) are indicated on the right.
Fig 3. Phosphorylation of GST-Cip29 protein fragments in X. laevis egg extract.(A) X. laevis Cip29 amino acid sequence indicating potential phosphorylation sites as predicted using the protein phosphorylation prediction tools, NetPhos 2.0, PHOSIDA, KinasePhos 2.0 and Scansite. The number of times each residue is predicted as a candidate phospho-site is indicated by shading, as described in the key. Ser/Thr residues that are conserved between X. laevis Cip29 and other vertebrates are underlined. (B) Schematic representation of GST-Cip29 fragments. Five Cip29 protein fragments (F1-F5), encompassing the entire Cip29 protein sequence, were each expressed with an N-terminal GST tag to facilitate purification. (C) Purified GST-Cip29 fusion proteins, F1-F5, (5μg each) were incubated in egg extract supplemented with 32P-ATP, in the absence or presence of 50ng/μl AT70, (21°C, 1h), then the recombinant proteins were recovered and subject to SDS-PAGE. Proteins were visualised by Sypro Ruby staining. Premature termination products or degradation products are denoted with asterisks. (D) After staining and protein quantification, the SDS-PAGE gel from (C) was dried and exposed to a phosphorimager screen for 48h to determine 32P-incorporation. (E) 32P-incorporation is presented for each fragment, in the absence or presence of AT70, relative to the amount of protein per band. The data shown in the graphs correspond to the mean of three independent experiments, and error bars indicate the SD. Note the axis break and scale change on the y axis to accommodate the high incorporation level in F5, relative to F1-F4.
Fig 4. Damage-dependent phosphorylation of Cip29 occurs on a conserved Ser residue of fragment 3.(A) F3 (5μg) was incubated in egg extract with 32P-ATP in the absence or presence of 50ng/μl AT70 and 5mM caffeine (21°C, 1h). F3 was recovered from the extract and 32P-incorporation was determined for each condition. (B) 5μg each of F3 (WT), F3 containing the double mutation S98A/T100A or the single mutation S95, were incubated as in (A) and 32P-incorporation determined for each fragment. (C) 5μg of full length His10-Cip29 was incubated as in (A) and 32P-incorporation was determined. In each case, the data represent an average of three independent experiments where error bars indicate the SD. Significance of the observed differences was evaluated using Studentâs t test (*P 0.01â0.05; **P 0.001â0.01).
Fig 5. Damage-dependent phosphorylation of recombinant full length Cip29.(A) Western blot of full length His10-Cip29 (WT) (lane 1) alongside His10-Cip29 (WT and S95A) incubated in egg extract with or without 50ng/μl AT70 (21°C, 1h) and recovered on nickel NTA agarose. Samples were resolved by electrophoresis on an 8% SDS-PAGE gel containing 15μM Phos-tag before immunoblotting with the anti-His antibodies. A modified Cip29 isoform is denoted by *. (B) Quantification of signal intensity of the modified Cip29 isoform (*) relative to the total Cip29 signal in each lane, expressed as fold-increase for AT70-treated extract relative to untreated extract. The data represent an average of three independent experiments where error bars indicate the SD. Significance of the observed differences was evaluated using Studentâs t test (*P 0.01â0.05).
Fig 6. Analysis of DNA end joining in Cip29-depleted egg extract.(A) Sequencing gel analysis of DNA end joining in X. laevis egg extracts using a defined repair substrate. Linearized repair substrate was incubated at 1ng/μl in mock-depleted, Mre11-depleted or Cip29-depleted extract (21°C, 6h). Where indicated, DMSO was added to a final concentration of 0.4% and DNA-PKi (NU7441), dissolved in DMSO, was added to a final concentration of 8μM. Cip29-depletion was performed with two different α-Cip29 antibodies (ÎCip29-1 and ÎCip29-2). Following incubation, plasmid DNA was recovered, digested with TaqαI and BstXI, resolved on a 20% denaturing polyacrylamide gel and exposed to a phosphorimager screen. Linearized substrate added to mock-depleted extract and processed immediately serves as an unrepaired control (0h). (B) Western blot of immunodepleted extracts from (A) indicating the depletion efficiency for Cip29 with both α-Cip29 antibodies. Uhrf1 serves as a loading control. Molecular weight markers (kDa) are indicated on the right.
Adamson,
A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response.
2012, Pubmed
Adamson,
A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response.
2012,
Pubmed
Aguilera,
Genome instability: a mechanistic view of its causes and consequences.
2008,
Pubmed
Anantha,
Requirement of heterogeneous nuclear ribonucleoprotein C for BRCA gene expression and homologous recombination.
2013,
Pubmed
Aravind,
SAP - a putative DNA-binding motif involved in chromosomal organization.
2000,
Pubmed
Bennetzen,
Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response.
2010,
Pubmed
Bensimon,
ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage.
2010,
Pubmed
Bensimon,
Beyond ATM: the protein kinase landscape of the DNA damage response.
2011,
Pubmed
Bhatia,
BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2.
2014,
Pubmed
Blom,
Sequence and structure-based prediction of eukaryotic protein phosphorylation sites.
1999,
Pubmed
Britton,
Cell nonhomologous end joining capacity controls SAF-A phosphorylation by DNA-PK in response to DNA double-strand breaks inducers.
2009,
Pubmed
Britton,
DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal.
2014,
Pubmed
Chen,
Accurate in vitro end joining of a DNA double strand break with partially cohesive 3'-overhangs and 3'-phosphoglycolate termini: effect of Ku on repair fidelity.
2001,
Pubmed
,
Xenbase
Choong,
An integrated approach in the discovery and characterization of a novel nuclear protein over-expressed in liver and pancreatic tumors.
2001,
Pubmed
Ciccia,
The DNA damage response: making it safe to play with knives.
2010,
Pubmed
Deng,
FUS is phosphorylated by DNA-PK and accumulates in the cytoplasm after DNA damage.
2014,
Pubmed
Domínguez-Sánchez,
Genome instability and transcription elongation impairment in human cells depleted of THO/TREX.
2011,
Pubmed
Dufu,
ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex.
2010,
Pubmed
Dutertre,
Cotranscriptional exon skipping in the genotoxic stress response.
2010,
Pubmed
Dutertre,
DNA damage: RNA-binding proteins protect from near and far.
2014,
Pubmed
Dutertre,
The emerging role of pre-messenger RNA splicing in stress responses: sending alternative messages and silent messengers.
2011,
Pubmed
Fan,
Global analysis of stress-regulated mRNA turnover by using cDNA arrays.
2002,
Pubmed
Felix,
A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity.
1989,
Pubmed
,
Xenbase
Fukuda,
Cloning and characterization of a proliferation-associated cytokine-inducible protein, CIP29.
2002,
Pubmed
Gardiner,
Identification and characterization of FUS/TLS as a new target of ATM.
2008,
Pubmed
Garner,
Studying the DNA damage response using in vitro model systems.
2009,
Pubmed
,
Xenbase
Gnad,
PHOSIDA 2011: the posttranslational modification database.
2011,
Pubmed
Göttlich,
Rejoining of DNA double-strand breaks in vitro by single-strand annealing.
1998,
Pubmed
,
Xenbase
Guan,
Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
1991,
Pubmed
Hanahan,
Hallmarks of cancer: the next generation.
2011,
Pubmed
Hocine,
RNA processing and export.
2010,
Pubmed
Izhar,
A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.
2015,
Pubmed
Jimeno,
Tho1, a novel hnRNP, and Sub2 provide alternative pathways for mRNP biogenesis in yeast THO mutants.
2006,
Pubmed
Jones,
XRad17 is required for the activation of XChk1 but not XCds1 during checkpoint signaling in Xenopus.
2003,
Pubmed
,
Xenbase
Jungmichel,
Proteome-wide identification of poly(ADP-Ribosyl)ation targets in different genotoxic stress responses.
2013,
Pubmed
Kim,
Substrate specificities and identification of putative substrates of ATM kinase family members.
1999,
Pubmed
Kinoshita,
Phosphate-binding tag, a new tool to visualize phosphorylated proteins.
2006,
Pubmed
Kleiman,
The BARD1-CstF-50 interaction links mRNA 3' end formation to DNA damage and tumor suppression.
2001,
Pubmed
Krietsch,
PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks.
2012,
Pubmed
Kumagai,
Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.
2000,
Pubmed
,
Xenbase
Labhart,
Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts.
1999,
Pubmed
,
Xenbase
Lackner,
A siRNA-based screen for genes involved in chromosome end protection.
2011,
Pubmed
Leaw,
Hcc-1 is a novel component of the nuclear matrix with growth inhibitory function.
2004,
Pubmed
Li,
A role for DEAD box 1 at DNA double-strand breaks.
2008,
Pubmed
Maréchal,
DNA damage sensing by the ATM and ATR kinases.
2013,
Pubmed
Mastrocola,
The RNA-binding protein fused in sarcoma (FUS) functions downstream of poly(ADP-ribose) polymerase (PARP) in response to DNA damage.
2013,
Pubmed
Matsuoka,
ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.
2007,
Pubmed
Mazan-Mamczarz,
RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation.
2003,
Pubmed
Obenauer,
Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs.
2003,
Pubmed
O'Neill,
Utilization of oriented peptide libraries to identify substrate motifs selected by ATM.
2000,
Pubmed
Paronetto,
The Ewing sarcoma protein regulates DNA damage-induced alternative splicing.
2011,
Pubmed
Paulsen,
A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability.
2009,
Pubmed
Polo,
Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair.
2012,
Pubmed
Rajesh,
The splicing-factor related protein SFPQ/PSF interacts with RAD51D and is necessary for homology-directed repair and sister chromatid cohesion.
2011,
Pubmed
Salton,
Involvement of Matrin 3 and SFPQ/NONO in the DNA damage response.
2010,
Pubmed
Smith,
An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63.
2009,
Pubmed
,
Xenbase
Smolka,
Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases.
2007,
Pubmed
Taylor,
DNA replication stress and cancer: cause or cure?
2016,
Pubmed
Taylor,
Depletion of Uhrf1 inhibits chromosomal DNA replication in Xenopus egg extracts.
2013,
Pubmed
,
Xenbase
Taylor,
The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis.
2010,
Pubmed
,
Xenbase
Wickramasinghe,
RNA Processing and Genome Stability: Cause and Consequence.
2016,
Pubmed
Wong,
KinasePhos 2.0: a web server for identifying protein kinase-specific phosphorylation sites based on sequences and coupling patterns.
2007,
Pubmed
Yamazaki,
The closely related RNA helicases, UAP56 and URH49, preferentially form distinct mRNA export machineries and coordinately regulate mitotic progression.
2010,
Pubmed
