LPS stimulated murine RAW264.7 cells, incubated with DMSO carrier or mycolactone
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE103002 | GEO | 2018/03/21
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 μg/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.
2010-05-09 | GSE20277 | GEO
Project description:We treated Jurkat cells for 48 hr with a sublethal dose of FK866 (5 nM) and DMSO (Mock, control treatment). RNA samples for microarrays derived from fractionated samples by sucrose gradient (sub-polysomes, polysomes), giving us the chance to perform an analysis among polysomal/subpolysomal distribution in treated or untreated cells and the possibility to identify the multi-level gene expression regulation effects of FK866. We are interested to find differentially expressed genes, in the early phase of cell response to FK866, and genes that account for a specific post-transcriptional regulation exerted by the cell in response to the drug. Keywords: polysomal profiling, translatome profiling, polysomal RNA, subpolysomal RNA, translational profiling, polysome profiling, post-transcriptional regulation, FK866, translational efficiency. Gene expression signals derived from the polysomal and subpolysomal RNA populations were compared by microarrays analysis to those obtained from total RNAs (derived from the sum of all the fractions in the polysomal gradient). Polysomal RNA, subpolysomal RNA and total RNA were isolated from Jurkat cells treated with FK866 5 nM or DMSO (mock, control treatment) for 48 hr. Cells lysates were collected from control cells (mock) and from treated cells (FK866). All experiments were run in biological quadruplicates.
2015-04-01 | E-GEOD-53772 | biostudies-arrayexpress
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 μg/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.
2010-05-27 | E-GEOD-20277 | biostudies-arrayexpress
Project description:We treated Jurkat cells for 48 hr with a sublethal dose of FK866 (5 nM) and DMSO (Mock, control treatment). RNA samples for microarrays derived from fractionated samples by sucrose gradient (sub-polysomes, polysomes), giving us the chance to perform an analysis among polysomal/subpolysomal distribution in treated or untreated cells and the possibility to identify the multi-level gene expression regulation effects of FK866. We are interested to find differentially expressed genes, in the early phase of cell response to FK866, and genes that account for a specific post-transcriptional regulation exerted by the cell in response to the drug. Keywords: polysomal profiling, translatome profiling, polysomal RNA, subpolysomal RNA, translational profiling, polysome profiling, post-transcriptional regulation, FK866, translational efficiency.
2015-04-01 | GSE53772 | GEO
Project description: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 Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.
2014-04-17 | E-GEOD-50650 | biostudies-arrayexpress
Project description:Sucrose gradient ultracentrifugation followed by microarray analysis was used to identify translationally-regulated mRNAs (mRNAs associated with ribosomes) from JFH1-infected and uninfected Huh-7.5.1 cells. Huh7 cells were infected with HCV JFH-1 strain. Controls consisted in non-infected cells and infected cells treated with an anti-protease. 3 days after infection, cell lysates were subjected to sucrose-gradient centrifugation in order to isolate polysome-free RNAs from polysome-bound (translated) RNAs. The non-polysomal, polysomal as well as cytosolic (non fractionned) RNAs, from infected, non-infected, and infected-treated cells were subject sur microarray analysis.
2013-02-12 | E-GEOD-44210 | biostudies-arrayexpress
Project description:We followed the polysomal association of maternal and early zygotic transcriptome over the first few hours of embryonic development, prior to and after MBT. We isolated polysome-associated (bound) and non-polysome-associated (unbound) mRNAs using sucrose gradient centrifugation followed by size fractionation. Using next generation sequencing (RNA-seq), we profiled the transcriptome in polysome-bound and unbound fractions. Our analysis revealed distinct dynamics of polysome association of cytoplasmically polyadenylated maternal mRNAs.
2017-10-14 | GSE88776 | GEO
Project description:During the peri-partum period, the lung must respond to many factors with potential to impact protein synthesis via regulation of translation initiation. Microarray analysis of polysomal versus total RNA from fetal day (FD) 19, FD22 and postnatal day 1 (P1) rat lungs was used to identify genes whose association with large polysomes changed either pre- or postnatally. Experiment Overall Design: Using three independent litters at each developmental time point, we prepared lysates from FD19, FD22 and P1 (5 h after birth) rat lungs (total 9 lysates). An aliquot of each lysate was used to prepare total RNA. Remaining lysate was subjected to sucrose density gradient centrifugation to isolate large polysomes ( n>7 ribosomes/mRNA) from which we isolated polysomal RNA. Matched total and polysomal RNAs were hybridized to Affymetrix expression arrays (total 18 chips).
2009-07-11 | E-GEOD-12153 | biostudies-arrayexpress
Project description:This experiment investigates the effect of mild cold-shock (32 degrees C, 24 hours) on the translatome of human HEK293 cells. After cold-shock, cells were treated with cycloheximide to block elongation, then sucrose density gradients were used to separate post-nuclear extracts into polysome and subpolysome fractions. The pooled polysomal fraction was labelled with Cy5, the pooled subpolysomal fraction was labelled with Cy3, and both were hybridised to the same array.
2017-02-23 | E-MTAB-5437 | biostudies-arrayexpress
Project description:We have performed sucrose-gradient-based isolation of polysomal fractions from untreated and TGF-beta treated MCF-10A and MCF7 cells, subjected these fractions to RNA-seq, and also sequenced total mRNA from each cell line in the treated and untreated condition
2016-09-24 | GSE81955 | GEO