po 077 identification of dhx30 as an inhibitor of the translation of pro apoptotic mrnas after p53 activation by nutlin

Introduction The transcription factor p53 can be efficiently activated by the small molecule Nutlin-3 without inducing genotoxic stress. Treatment of different cell lines with this small molecule can result in different phenotypes, ranging from cell cycle arrest to apoptosis. HCT116 (colon cancer-derived cells) and SJSA1 (osteosarcoma-derived cells) were used to model the opposite behaviour respectively, by analysing the transcriptional and translational responses after Nutlin-3 treatment. Material and methods Total and polysomal-bound mRNAs were collected and sequenced after 12 hour of 10 uM Nutlin-3 treatment. A bioinformatics analysis of the polysome-enriched mRNAs using Weeder allowed the identification of a 3'UTR motif ('CG-rich') which is enriched in the translationally upregulated genes of SJSA1. The effect of the motif on translation was evaluated after cloning its consensus sequence in the 3'UTR of the b-globin gene, which was put downstream the luciferase reporter. The activity of the construct was evaluated after 12 or 24 hours of Nutlin-3. The same consensus was used for a pull-down experiment followed by mass spectrometry to identify proteins interacting with it. Results and discussions RNA-seq data indicate that HCT116 and SJSA1, although sharing almost completely the transcriptional program lead by p53, show almost no overlap at a translation level. SJSA1 present different pro-apoptotic translationally-upregulated genes after Nutlin-3, which have one or more instances of a CG-rich motif in the 3'UTR. The impact of the motif is to enhance the activity of the luciferase reported when cloned in two copies flanking the 3'UTR of the b-globin gene, but only in SJSA1. A pull-down experiment using an RNA bait with the consensus motif was used to identify interactors, among which DHX30 was deeply studied. DHX30 silencing in HCT116 causes: 1) enhanced the activity of the reporter construct after Nutlin; 2) polysomal association of selected mRNAs containing the motif; 3) induction of apoptosis as assessed by Annexin-V staining. In addition, silencing of DHX30 in U2OS cells decreased their survival after Nutlin-3 treatment. Conclusion We show how a p53-dependent transcriptional program can be shaped at a translational level thanks to the action of a CG-rich motif which is enriched in the 3'UTR of some pro-apoptotic mRNAs and that can be bound by DHX30. This protein acts as a translational repressor of mRNAs containing the motif. The exact mechanism and the generalisation of the model are currently being investigated

The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus

Polo-like kinases (Plk1-4) are emerging as an important class of proteins involved in many aspects of cell cycle regulation and response to DNA damage. Here, we report the cloning of a fifth member of the polo-like kinase family named Plk5. DNA and protein sequence analyses show that Plk5 shares more similarities with Plk2 and Plk3 than with Plk1 and Plk4. Consistent with this observation, we show that mouse Plk5 is a DNA damage inducible gene. Mouse Plk5 protein localizes predominantly to the nucleolus, and deletion of a putative nucleolus localization signal (NoLS) within its N-terminal moiety disrupts its nucleolar localization. Ectopic expression of Plk5 leads to cell cycle arrest in G1, decreased DNA synthesis, and to apoptosis, a characteristic it shares with Plk3. Interestingly, in contrast to mouse Plk5 gene, the sequence of human Plk5 contains a stop codon that produces a truncated protein lacking part of the kinase domain

The DNA damage effector Chk1 kinase regulates Cdc14B nucleolar shuttling during cell cycle progression

Chk1 is a critical effector of DNA damage checkpoints necessary for the maintenance of chromosome integrity during cell cycle progression. Here we report, that Chk1 co-localized with the nucleolar marker, fibrillarin in response to radiation-induced DNA damage in human cells. Interestingly, in vitro studies using GST pull down assays identified the dual-specificity serine/threonine nucleolar phosphatase Cdc14B as a Chk1 substrate. Furthermore, Chk1, but not a kinase-dead Chk1 control, was shown to phosphorylate Cdc14B using an in vitro kinase assay. Co-immunoprecipitation experiments using exogenous Cdc14B transfected into human cells confirmed the interaction of Cdc14B and Chk1 during cell cycle. In addition, reduction of Chk1 levels via siRNA or UCN-01 treatment demonstrated that Chk1 activation following DNA damage was required for Cdc14B export from the nucleolus. these studies have revealed a novel interplay between Chk1 kinase and Cdc14B phosphatase involving radiation-induced nucleolar shuttling to facilitate error-free cell cycle progression and prevent genomic instability