Polysome-associated mRNA profiling of cancer cells in response to CXCL12 and IGF1
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Zhang XH
PROVIDER: S-ECPF-GEOD-43296 | biostudies-other | 2013 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:CXCL12 and IGF1 are key secreting molecules produced by cancer-associated fibroblasts in breast cancer. These factors promote the survival of disseminated cancer cells in the bone marrow. To assess the combined responses elicited by CXCL12 and IGF1, we examined the translating transcriptome of cancer cells in response to these two factors by Translating Ribosome Affinity Purification (TRAP)-RNAseq.
2013-11-01 | GSE43296 | GEO
Project description:CXCL12 and IGF1 are key secreting molecules produced by cancer-associated fibroblasts in breast cancer. These factors promote the survival of disseminated cancer cells in the bone marrow. To assess the combined responses elicited by CXCL12 and IGF1, we examined the translating transcriptome of cancer cells in response to these two factors by Translating Ribosome Affinity Purification (TRAP)-RNAseq. MDA-MB-231 cells were engineered to express an EGFP-tagged version of ribosomal protein L10a. This allows the retrieval of polysome-associated mRNA by anti-GFP pull down (TRAP) and profiling the translating transcriptome by RNAseq. EGFP-L10a+ cancer cells were serum starved (0.2% serum) for 24 hours, and then treated with CXCL12 (30ng/mL) + IGF1 (10ng/mL) or CXCL12 (300ng/mL) + IGF1 (100ng/mL) for 6hrs. Two biological replicates were profiled for each condition.
2013-11-01 | E-GEOD-43296 | biostudies-arrayexpress
Project description:Infection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation while viral translation proceeds efficiently. VSV RNA synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces 5 mRNAs that are structurally indistinct to cellular mRNAs with respect to their 5' cap-structure and 3'-polyadenylate tail. Using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mRNA, we interrogate the impact of VSV infection of HeLa cells on translation. Analysis of sequence reads in the different fractions shows >60% of total cytoplasmic and polysome-associated reads map to the 5 viral genes by 6 hours post-infection, a time point at which robust host cell translational shut-off is observed. Consistent with an overwhelming abundance of viral mRNA in the polysome fraction, the reads mapping to cellular genes were reduced. The cellular mRNAs that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more AU rich, features that are shared with the viral mRNAs. Several of those mRNAs encode proteins known to positively affect viral replication, and using chemical inhibition and siRNA depletion we confirm that the host chaperone heat shock protein 90 (hsp90) and eukaryotic translation initiation factor 3A (eIF3A)-encoded by 2 such mRNAs-support viral replication. Correspondingly, regulated in development and DNA damage 1 (Redd1) encoded by a host mRNA with reduced polysome association inhibits viral infection. These data underscore the importance of viral mRNA abundance in the shut-off of host translation in VSV infected cells and link the differential translatability of some cellular mRNAs with pro- or antiviral function.
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Project description:Polysome-profiling is commonly used to study translatomes and applies laborious extraction of efficiently translated mRNA (associated with >3 ribosomes) from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for efficiently translated mRNA in only one or two fractions, thereby reducing sample handling 5-10-fold. The technique generates polysome-associated RNA with a quality reflecting the starting material and, when coupled with smart-seq2 single-cell RNA sequencing, translatomes in small tissues from biobanks can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associates with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in biobanks.
| S-EPMC5758873 | biostudies-literature
Project description:During the past decade, there has been growing interest in the role of translational regulation of gene expression in many organisms. Polysome profiling has been developed to infer the translational status of a specific mRNA species or to analyze the translatome, i.e. the subset of mRNAs actively translated in a cell. Polysome profiling is especially suitable for emergent model organisms for which genomic data are limited. In this paper, we describe an optimized protocol for the purification of sea urchin polysomes and highlight the critical steps involved in polysome purification. We applied this protocol to obtain experimental results on translational regulation of mRNAs following fertilization. Our protocol should prove useful for integrating the study of the role of translational regulation in gene regulatory networks in any biological model. In addition, we demonstrate how to carry out high-throughput processing of polysome gradient fractions, for the simultaneous screening of multiple biological conditions and large-scale preparation of samples for next-generation sequencing.
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Project description:[This corrects the article PMC5431591.].
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Project description:Changes in polysome-bound mRNA (translatome) are correlated closely with changes in the proteome in cells. Therefore, to better understand the processes mediating the response of glioblastoma to ionizing radiation (IR), we used polysome profiling to define the IR-induced translatomes of a set of human glioblastoma stem-like cell (GSC) lines. Although cell line specificity accounted for the largest proportion of genes within each translatome, there were also genes that were common to the GSC lines. In particular, analyses of the IR-induced common translatome identified components of the DNA damage response, consistent with a role for the translational control of gene expression in cellular radioresponse. Moreover, translatome analyses suggested that IR enhanced cap-dependent translation processes, an effect corroborated by the finding of increased eIF4F-cap complex formation detected after irradiation in all GSC lines. Translatome analyses also predicted that Golgi function was affected by IR. Accordingly, Golgi dispersal was detected after irradiation of each of the GSC lines. In addition to the common responses seen, translatome analyses predicted cell line-specific changes in mitochondria, as substantiated by changes in mitochondrial mass and DNA content. Together, these results suggest that analysis of radiation-induced translatomes can provide new molecular insights concerning the radiation response of cancer cells. More specifically, they suggest that the translational control of gene expression may provide a source of molecular targets for glioblastoma radiosensitization. Cancer Res; 76(10); 3078-87. ©2016 AACR.
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Project description:The environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) causes a multitude of human illnesses. In order to more fully understand the underlying biology of TCDD toxicity, we tested the hypothesis that new candidate genes could be identified using polysome RNA from TCDD-treated mouse Hepa-1c1c7 cells. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for the remaining polysome RNAs, levels are regulated via several different mechanisms, including a "tagging" of mRNAs in the nucleus for immediate polysome entry; and (iv) most importantly, a gene list derived from differentially expressed polysome RNA generated new genes and cell pathways potentially related to TCDD biology.
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Project description:In the malaria parasite Plasmodium falciparum, global studies of translational regulation have been hampered by the inability to isolate malaria polysomes. We describe here a novel method for polysome profiling in P. falciparum, a powerful approach which allows both a global view of translation and the measurement of ribosomal loading and density for specific mRNAs. Simultaneous lysis of infected erythrocytes and parasites releases stable, intact malaria polysomes, which are then purified by centrifugation through a sucrose cushion. The polysomes are resuspended, separated by velocity sedimentation and then fractionated, yielding a characteristic polysome profile reflecting the global level of translational activity in the parasite. RNA isolated from specific fractions can be used to determine the density of ribosomes loaded onto a particular transcript of interest, and is free of host ribosome contamination. Thus, our approach opens translational regulation in malaria to genome-wide analysis.
| S-EPMC3135974 | biostudies-literature
Project description:An important but often overlooked aspect of gene regulation occurs at the level of protein translation. Many genes are regulated not only by transcription but by their propensity to be recruited to actively translating ribosomes (polysomes). Polysome profiling allows for the separation of unbound 40S and 60S subunits, 80S monosomes, and actively translating mRNA bound by two or more ribosomes. Thus, this technique allows for actively translated mRNA to be isolated. Transcript abundance can then be compared between actively translated mRNA and all mRNA present in a sample to identify instances of post-transcriptional regulation. Additionally, polysome profiling can be used as a readout of global translation rates by quantifying the proportion of actively translating ribosomes within a sample. Previously established protocols for polysome profiling rely on the absorbance of RNA to visualize the presence of polysomes within the fractions. However, with the advent of flow cells capable of detecting fluorescence, the association of fluorescently tagged proteins with polysomes can be detected and quantified in addition to the absorbance of RNA. This protocol provides detailed instructions on how to perform fluorescent polysome profiling in C. elegans to collect actively translated mRNA, to quantify changes in global translation, and to detect ribosomal binding partners.
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