Date Published: July 17, 2017
Publisher: Public Library of Science
Author(s): Janet Holden, Elaine M. Taylor, Howard D. Lindsay, Sue Cotterill.
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.
A hallmark of cancer, genome instability is recognised as a key driving force in cancer progression, since it facilitates the acquisition of further tumourigenic changes . Genome instability may result from exposure to DNA damaging agents, exogenous or endogenous in origin, or as a consequence of the replicative stress which generally accompanies oncogenic transformation [2, 3]. A variety of DNA damage response (DDR) pathways exist to contend with such genetic damage. Principal amongst these is DNA damage signalling via the phosphatidylinositol 3-kinase-like kinases (PI3KK), ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related), which elicits a variety of cellular responses, including activation of DNA repair mechanisms, cell cycle arrest or apoptosis . These outcomes are achieved through phosphorylation-dependent modulation of protein function and also as a result of altered gene expression, by activation of DNA damage-inducible transcription, and through post-transcriptional changes in mRNA processing and translation. Of these, RNA processing has only relatively recently started to be recognised as an important element of the DDR [5, 6].
The use of animals (Xenopus laevis) in this work was approved by the Lancaster University Ethical Review Process (ERP) and is covered by Home Office Project Licence 7007392.
RNA processing is increasingly being recognised as an important element in the maintenance of genome integrity since it helps both to prevent genetic damage, through the avoidance of cotranscriptional R-loop formation, and to promote effective DNA damage responses, through regulated expression of various DDR proteins [5, 6]. Moreover, a growing list of RNA processing factors has been identified that are either recruited to sites of DNA damage, or which interact with known DNA repair factors, suggesting that they may play a direct role in DNA damage detection, signalling and/or repair [19–21, 50–52]. Consistent with the involvement of RNA processing factors in diverse aspects of the DNA damage response, a number of RBPs have been identified as targets of DNA damage-dependent signalling.
Our data demonstrate that X. laevis Cip29, a putative RNA export factor, is rapidly phosphorylated in response to DNA double-strand breaks in Xenopus egg extracts. This DNA damage-inducible phosphorylation is dependent on the ATM checkpoint kinase and we have identified the damage-dependent phosphorylation site as a conserved serine residue, Ser95. We have determined that Cip29 is not required for efficient DNA end joining repair in X. laevis egg extracts and suggest that the damage-dependent modification of Cip29 may instead relate to a role in regulation of gene expression after DNA damage.