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Global analysis of the actively translating portion of the transcriptome of control and YBX1 depleted proliferating epidermal cells by high-resolution RNA-seq analysis

ABSTRACT: To investigate the mechanisms underlying the effects of YBX1 depletion on epidermal progenitors, we performed deep RNA-seq analysis of control versus YBX1-depleted proliferating epidermal cells. Since YBX1 can affect both transcription and translation, we analyzed both total mRNA and polysomaly associated mRNAs using sucrose density gradient sedimentation. Deep RNA-seq analysis based on duplicate samples showed that 57 genes were significantly modulated in YBX1 siRNA treated cells at the level of total mRNA as compared to control siRNA cells, while 115 genes exhibited changes in polysomal mRNA, respectively. Considering the predominantly cytosolic localization of YBX1 in the epidermis, we hypothesized that most of the observed transcriptional changes are secondary to changes in YBX1 mediated translational regulation of specific mRNA subsets. To identify such translational targets of YBX1, we analyzed transcripts that were equally expressed within total RNA populations of control and YBX1 siRNA cells but were selectively modulated in the polysomal mRNA fractions of YBX1 depleted cells. Further examination revealed that out of the 96 affected polysomaly associated mRNAs, 39 were most discriminatory in expression in polysomal versus total mRNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE110014 | GEO | 2019/05/28

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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Global analysis of the actively translating portion of the transcriptome of control and YBX1 depleted proliferating epidermal cells by high-resolution RNA-seq analysis

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