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Transcriptome and translatome profiling of MCF7 vector cells after p53 activation

ABSTRACT: Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a

ORGANISM(S): Homo sapiens

PROVIDER: GSE50650 | GEO | 2014/04/17

SECONDARY ACCESSION(S): PRJNA218203

REPOSITORIES: GEO

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Project description:Translatome analysis by sucrose gradient centrifugation of cell lysates followed by microarray profiling of the polysomal and subpolysomal RNA fractions represents a way of both studying translational control networks and better approximating the proteomic representation of cells. It is an established notion that translational control takes place essentially at the translation initiation level, therefore the variation in abundance of a given mRNA species on polysomes can be directly related to the variation in abundance of the corresponding protein. Comparison of translatome profile changes with corresponding transcriptome profile changes can provide a measure of the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. To provide a direct experimental evaluation of the phenomenon, we decided to study a classical example of transcriptional reprogramming of gene expression: Epidermal Growth Factor (EGF) treatment. This stimulus triggers a well known chain of intracellular transduction events, ultimately resulting in a multifaceted phenotypic spectrum of changes with prevalent induction of cell growth and proliferation. We subjected HeLa cells to serum starvation for 12h and then we added EGF at final concentration of 1 Î¼g/ml, profiling before and after 40 minutes of treatment the transcriptome, the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation, and also the mRNA content of the subpolysomal pool, expected not to be actively translated. Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, EGF starvation release. The comparison between transcriptional and polysomal profiling was used for the discovery of general and mRNA-specific changes in the translation state of the serum starved HeLa cells transcriptome in response to EGF stimulus. To identify translationally regulated mRNA molecules, gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from unfractionated total RNAs. Polysomal RNA, subpolysomal RNA and total RNA were isolated from HeLa cells serum starved and treated with EGF. Cells lysates were collected before (t = 0 min) and after (t = 40 min) EGF treatment. All experiments were run in triplicates.

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Transcriptome and translatome profiling of MCF7 vector cells after p53 activation

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