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.
Project description:This study is complementary to the "Genome-wide analysis of host mRNA translation during hepatitis C virus infection" study (GSE44210), for which 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. MicroRNA arrays have been conducted in parallel on total RNA from infected and uninfected cells, in order to determine if microRNA regulation could explain some of the mRNA translation regulations. Huh7 cells were infected with HCV JFH-1 strain. Controls consisted in non-infected cells. 3 days after infection, total RNAs were extracted from cell lysate and subjected to miRNA microarray analysis.
Project description:eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the requirement for eIF4E dose at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we surprisingly found that 50% reduction in eIF4E, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation and tumorigenesis. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for eIF4E dose specifically in translating a network of mRNAs enriched for a unique 5’UTR signature. In particular, we demonstrate that eIF4E dose is essential for translating mRNAs regulating reactive oxygen species (ROS) that fuel transformation and cancer cell survival in vivo. Therefore, mammalian cells have evolved surplus eIF4E levels that cancer cells hijack to drive a translational program supporting tumorigenesis Total cellular RNA and high MW polysome associated RNA were isolated from matched untransformed and transformed WT and Eif4e+/- MEFs for analysis on Affymetrix Mouse Gene 1.0 ST arrays. The difference in log2 RMA intensity between matched polysomal RNA and total RNA was taken to quantify translational efficiency (TE).
Project description:Type I interferons are critical anti-viral cytokines during virus infections and have also been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The secretion of type I interferon of pDCs is modulated by Siglec-H, a DAP12 associated receptor on pDCs. We showed that Siglec-H deficient pDCs produce more of the type I interferon IFN-α in vitro and that Siglec-H ko mice produce more IFN-α after murine cytomegalovirus (mCMV) infection in vivo, leading to efficient clearance of the virus. Furthermore, ageing Siglec-H ko mice showed a mild form of systemic autoimmunity. In contrast, Siglec-H ko mice developed a severe form of systemic lupus-like autoimmune disease with strong kidney nephritis several weeks after a single mCMV infection. This induction of systemic autoimmune disease after virus infection in Siglec-H ko mice was accompanied by a type I interferon signature and fully dependent on type I interferon signaling. These results show that Siglec-H normally serves as modulator of type I interferon responses after infection with a persistent virus and thereby prevents induction of autoimmune disease. For microarray experiments gene expression profiles of total splenic cells from two wt and Siglec-H ko mice 26 weeks after infection with luciferase expressing murine Cytomegalovirus (5x105 pfu) or from two uninfected wt and Siglec-H ko control mice were analyzed
Project description:Growth phase dependent translational regulation of Hf. volcanii was analyzed on a genome-wide scale. The relative amounts of free versus polysome-bound mRNA (separated by gradient centrifugation) were quantified in exponential as well as stationary growth phase by the use of shotgun DNA microarrays.
Project description:Rationale: Low B12 has been shown to play an important role in the prediction of metabolic risk, but its significance and mechanism in the development of adiposity and adipose tissue dysfunction is largely unknown. Objective: To investigate the role of B12 and folic acid in the development of adipocyte dysfunction. Methods and Results: Microarray analysis of human adipocytes (CHUB-S7 cell line) cultured and differentiated in customised media with varying concentrations of B12 and folic acid led to the identification of two important pathways: cholesterol synthesis and unfolded protein response (UPR). Adipocytes cultured in media with low B12 (150 pmol/L) or no B12 had increased intracellular total cholesterol, higher secreted homocysteine levels, induced UPR and reduced glucose uptake capacity compared to adipocytes cultured in normal media with higher B12. The folate concentrations had either no or little effect on the measured functions. Further analysis of these adipocytes for overall DNA methylation showed that the promoter regions of sterol regulatory element-binding transcription factor 1 (SREBF1) and low density lipoprotein receptor (LDLR) were hypomethylated in the low and no B12 conditions. The SREB proteins (SREBP1 and 2) and mRNA expressions (SREBF1 and LDLR) were also increased in the same conditions. Conclusion: The data suggest that low B12 can lead to adipocyte dysfunction by inducing excess cholesterol biosynthesis, homocysteine production and induction of UPR and overall adipocyte dysfunction. Both of these pathways and adipocyte dysfunction play a significant role in the development of cardiovascular diseases. Independent replicate samples of the human adipocyte cell line CHUB-S7 were treated with four different concentrations of B12 and folate.
Project description:We investigated genome-wide changes in mRNA translation in Arabidopsis thaliana T87 suspension cell cultures which thought to be one of the host materials for bioreactor. Global translational repression was observed in cells of 8 day after inoculation that is thought to be stressful condition by the nutrient deficiency and hypoxia. This suggested the negative effect of the global translational repression on transgene expression. On the other hand, previous study using heat stress showed that some mRNAs were actively translated under such stressful condition, suggesting the existence of mRNA that were actively translated in cells of 8 day after inoculations. To identify mRNAs that escape global translational repression on 8 day and its cis-elements would be the 1st step to make the system for higher transgene expression by the escaping global translational repression. To this end, we subjected polysomal RNA and non-polysomal RNA from sucrose gradient fractionated cell lysates to the co-hybridization on Agilent Arabidopsis 4 Oligo Microarrays. The ratio of signal intensities (polysomal RNA: total RNA) was used as an indicator of the translation state for each transcript. Experiment using two-fractionated mRNA, Polysomal mRNA vs. total mRNA. Biological replicates: 1
Project description:Transriptional profiling of white adipose tissue extracted from obese mice. White adipose tissues were extrated from Oma1-deficient mouse and control after 20 weeks of diet-induced obesity. RNA were extracted and hybridated with Mouse Gene 1.0 ST from Affymetrix microarrays.
Project description:Adamts12-deficient mice undergo more severe colitis than WT mice after induction with DSS. We used microarrays to determine the gene expression differences between Adamts12-deficient and WT mice during ulcerative colitis induced with DSS (dextran sodium sulfate) Fragments of distal colon from DSS-treated (2% DSS during 7 days and 1 day of recovery) and untreated Adamts12-deficient and WT mice were obtained for RNA extraction and hybridiztion with Affymetrix microarrays
Project description:Listeria monocytogenes is a facultative intracellular bacterial pathogen that tightly regulates the activities of various virulence factors during infection. A mutant strain (the plcBM-NM-^Tpro mutant) that has lost the ability to control the activity of a phospholipase C (PC-PLC) is attenuated a hundred fold in mice. This attenuation is not due to a lack of bacterial fitness, but appears to result from a modified host response to infection. The transcriptomic pattern of immunerelated genes in infected macrophages indicated no differential response to wild-type L. monocytogenes vs the plcBM-NM-^Tpro mutant. Cultures of bone marrow derived macrophages from BALB/c were infected with either wild type or mutant L. monocytogens for 3, 6, or 9 hrs. The macrophages were then collected and RNA isolated for microarray analysis of gene expression.