Translational control mechanisms in the p53 response network

Zaccara, Sara (2015) Translational control mechanisms in the p53 response network. PhD thesis, University of Trento.

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The sequence-specific transcription factor p53 is considered a master gene of cellular responses to homeostasis changes. It is also a prominent tumor suppressor gene with the title of “guardian of the genome”. The increasing number of transcriptome analyses in cell lines treated with different agents activating p53, continues to add complexity to the vast transcriptional networks p53 regulates. To investigate mRNA translational control as an additional dimension of p53-directed gene expression responses, we performed translatome analyses upon its activation either by different agents or cellular contexts. Considered as a proxy for the proteome, the translatome allows us to characterize the translational status of each mRNA, independently from transcriptional modulations, and to evaluate the implications or correlations of changes in relative mRNA translation efficiencies with the phenotypic outcome. We first performed treatment-specific translatome profiling in MCF7 cells upon Doxorubicin and Nutlin-3a treatments. Among translated genes, we detected the presence of translationally enhanced mRNAs with a virtually absent transcriptional modulation; those genes were enriched for apoptotic functions, suggesting that the apoptotic phenotype might be controlled not only at the transcriptional, but also at the translational level. Seeking mechanisms underlying the mRNAs translational rate upon p53 activation, we identified the modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets, modulated at the translational level in a p53-dependent manner. In detail, we demonstrated the contribution of at least two p53-dependent translational mechanisms related to YBX1 translational repression, suggesting the presence of a controlled regulon at the crossroad of YBX1 mRNA translation. Given our finding that apoptotic genes appear to be controlled by p53 also at the translational level, we decided to explore whether mRNAs translational control mechanisms are indeed an additional checkpoint to the phenotype. To this aim, we performed a cell-type specific translatome study upon Nutlin-3a treatment, a drug with evident therapeutic prospective. SJSA1, HCT116 and MCF7 cells were chosen as they exhibit different cellular responses to Nutlin-3A (cell cycle arrest, apoptosis, or both, respectively). Our preliminary data suggests that translational modulation can affect the complex process of cell fate choice upon p53 activation. Indeed, a lack of overlap among genes differentially modulated at the translational level was evident. Motif search analysis at the 5’- and 3’-UTR of those genes highlighted the presence of different motifs in the three cell lines and the specific correlation of a C-rich motif with the apoptotic phenotype. Preliminary data on this motif will be presented and discussed. Two independent projects will be presented as appendixes, both of them related to the general idea that more than one factor may determine the p53 response. Starting from the analysis of possible p53 interactions with other transcriptional co-factors, we investigated the cooperative interaction between p53 and NFκB. For the second project, combining data previously obtained by means of yeast-based p53 transactivation assays, we developed an algorithm, p53retriever, to scan DNA sequences and thus identify p53 response elements and classify them based on their transactivation potential.

Item Type:Doctoral Thesis (PhD)
Doctoral School:Biomolecular Sciences
PhD Cycle:27
Subjects:Area 05 - Scienze biologiche > BIO/11 BIOLOGIA MOLECOLARE
Area 05 - Scienze biologiche > BIO/13 BIOLOGIA APPLICATA
Area 05 - Scienze biologiche > BIO/18 GENETICA
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