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Ribo-Seq experiments in HCT116 WT and TP53-/- cells upon neocarzinostatin treatment

ABSTRACT: The transcription factor p53 exerts its tumor suppressive effects through transcriptional activation of numerous target genes controlling cell cycle arrest, apoptosis, cellular senescence and DNA repair. In addition, there is evidence that p53 influences the translation of specific mRNAs, including translational attenuation of ribosomal protein synthesis and translational activation of MDM2. A challenge in the analysis of translational control is that changes in mRNA abundance exert a kinetic (passive) effect on ribosome densities. In order to separate these passive effects from active regulation of translation efficiency in response to p53 activation, we conducted a comprehensive analysis of translational regulation by comparative analysis of mRNA levels and ribosome densities upon DNA damage induced by neocarzinostatin in wild-type and TP53-/- HCT116 colorectal carcinoma cells. Thereby, we identified a specific group of mRNAs whose translation is actively up-regulated in response to p53 activation, the majority of which are transcribed from p53 target genes including MDM2, SESN1 and CDKN1A. By subsequent polysome profile analysis, we could demonstrate that a p53-dependent switch in transcript isoform expression is responsible for translational activation of SESN1, whereas translational activation of CDKN1A occurs through a trans-acting mechanism that affects multiple transcript isoforms.

ORGANISM(S): Homo sapiens

PROVIDER: GSE166783 | GEO | 2022/04/13

REPOSITORIES: GEO

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Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2Î±, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2Î±/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2Î± S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2Î± phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2Î±, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.

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Ribo-Seq experiments in HCT116 WT and TP53-/- cells upon neocarzinostatin treatment

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