Unknown,Transcriptomics,Genomics,Proteomics

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Translatome and transcriptome profiling of SH-SY5Y cells treated with sublytic doses of staphylococcal alpha-hemolysin

ABSTRACT: Genome-wide analysis of translation has the potential to provide major contributions in understanding the pathophysiology of infection processes, given the complex interplay between pathogens and host cells. Informations about the translational state of mRNAs or the activity of RNA binding proteins and ncRNAs after treatment with sublytic doses of pore forming toxins are completely missing. This study uncovers the reshaping undergoing in the translational control system of the host in response to sublytic doses of staphylococcal α-hemolysin (AHL). Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, staphylococcal α-hemolysin, pore forming toxins, PTF. The comparison between translatome and transcriptome profiling was used to discover mRNA-specific changes of the SH-SY5Y cells transcriptome and translatome in response to sublytic doses of staphylococcal α-hemolysin (AHL). To identify translationally regulated mRNAs, gene expression signals derived from the polysomal mRNA populations were compared by microarrays analysis to those obtained from total RNAs. Polysomal mRNA and total mRNA were isolated from SH-SY5Y cells treated with sublytic doses (3nM) of AHL for 2 hours. Cells lysates were collected from untreated cells (control) and from treated cells. All experiments were run in biological triplicates.

ORGANISM(S): Homo sapiens

SUBMITTER: Toma Tebaldi

PROVIDER: E-GEOD-50652 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description:Neuroblastoma (NB) is the most frequent extracranial solid tumour of childhood. Clinical courses are highly variable, ranging from spontaneous regression/maturation to rapid progression despite intensive multimodal therapy. The estimation of 5-year event free survival in high-risk patients of only about 40 % stresses an importance of novel therapeutic strategies. A number of iron chelators have demonstrated marked in vitro and in vivo anti-tumor activity and are currently being developed as novel anti-cancer agents. Therefore, the potential antitumor effect of iron chelators in NB cancer was investigated. Among the compounds tested, ciclopirox olamine (CPX) was shown to be one of the most effective intracellular iron chelators in NB cells. To unveil the molecular mechanisms underlying the effects of CPX on viability of NB cells, microarray analysis was performed in CHP134 control cells and cells treated with 5 M-BM-5M CPX for 90 minutes. Inclusion of both total RNA (reflecting transcriptional status of the cells) and polysomal RNA (approximating the proteomic representation of the cells) provided us with a deeper understanding of changes in the cells upon CPX treatment. Keywords: ciclopirox olamine, iron chelator, neuroblastoma, translatome profiling, transcriptome profiling. Keywords: ciclopirox olamine, iron chelator, neuroblastoma, translatome profiling, transcriptome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling Microarray analysis was performed to enable a comprehensive view of the changes in the transcriptional and translational status of the cells in response to the treatment with an iron chelator CPX. Polysomal RNA and total RNA were isolated from vehicle-treated CHP134 cells and CHP134 cells treated with 5 M-BM-5M CPX for 90 minutes. Each condition is represented by three biological replicates, i.e. the experiment was repeated starting each time from cells seeding yielding an independent RNA sample. In total, 12 samples corresponding to 4 conditions were subjected to microarray analysis.

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Translatome and transcriptome profiling of SH-SY5Y cells treated with sublytic doses of staphylococcal alpha-hemolysin

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