Project description:The I/11 and R10 erythroid progenitor cell line was cultivated in serum free medium (StemPro34) supplemented with Epo (0,5U/ml), SCF (100ng/ml) and dexamethasone (10-6M). To identify genes specifically regulated by Epo/SCF-induced polysome recruitment, cells were factor deprived for 4h and subsequently treated for 2h with 5U/ml Epo and 200ng/ml SCF or left untreated. Subsequently we isolated both total and polysome bound mRNA from each condition, which was hybridised to oligonucleotide arrays (Affymetrix). Analysis of data with Rosetta Resolver allowed to identify and compare Epo/SCF induced gene expression in total and polysome bound RNA. To assess gene expression during erythroid differentiation, cells were induced to differentiate in presence of 5U/ml Epo and 0.5mg/ml Fe-loaded transferrin. Polysome bound mRNA was isolated from cells proliferating in presence of Epo, SCF and dexamethasone (renewal conditions), and from cells induced to differentiate for 48h or 60h.
Project description:We investigated mRNA regulatory proteins of the ELAV family following 10 min global brain ischemia and 8 hrs reperfusion (8R) or non-ischemic controls (NIC) in rat hippocampal CA1 and CA3. There were three main experiments: (1) Shot-gun proteomics of polysome pellets from NIC and 8R CA1 and CA3, (2) Shot-gun proteomics of ELAV immunoprecipitate eluents from NIC and 8R CA1 and CA3, and (3) Proteomics of ELAV antisera-reactive bands excised from nitrocellulose following ELAV Western blot.
Project description:Translational control is a key regulatory step in the expression of genes as proteins. In plant cells, translational efficiency of mRNAs differs on different mRNA species, and the efficiency dynamically changes in various conditions. To gain a global view of translational control throughout growth and development, we performed genome-wide analysis of polysome association of mRNA over growth and leaf development in Arabidopsis thaliana by applying the mRNAs in polysome to DNA microarray. This analysis revealed that the degree of polysome association of mRNA had different levels depending on mRNA species, and the polysome association changed greatly throughout growth and development for each. In the growth stage, transcripts showed varying changes in polysome association from strongly depressed to unchanged degree, with the majority of transcripts showing dissociation from ribosomes. On the other hand, during leaf development, the polysome association of transcripts showed a normal distribution from repressed to activated mRNAs when comparing between expanding and expanded leaves. In addition, functional category analysis of the microarray data suggested that translational control has a physiological significance in plant growth and development process, especially in category of signaling and protein synthesis. Besides this, we compared changes in polysome association of mRNAs between various conditions and characterized translational controls in each. This result suggested that mRNAs translation might be controlled by complicated mechanisms for response to each condition. Our results highlight the importance of dynamic changes in mRNA translation in plant development and growth. Experiment using two-flactionated mRNA in 4 developmental stages, Polysomal mRNA vs. total mRNA. Biological replicates: 2. Compared 2DAG and 21DAG, or Young leaves and Mature leaves.
Project description:The goal was to measure the expression and polysome association of genes with polysomes in human embryonic stem cells H9 human embryonic stem cells were kept in pluripotent conditions. Total RNA and polysome extracts were isolated.
Project description:The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO, underscore the relevance of GRTH in translation. Studies showed the association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. Microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT) and GRTH KO mice.
Project description:These are the ribosomal subunit fractions from the polysome gradients. investigating effect of heat shock on procyclic-form trypanosomes.