Expression & polysome association in pluripotent human embryonic stem cells
ABSTRACT: 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:Gene expression in the total RNA and heavy polysome fractions of Eif4g3 siRNA treated lymph node stromal cells (LNSCs) compared to control-sIRNA treated samples The objective of this study was to identify genes whose translation are reduced after silencing Eif4g3 (the gene which encodes the translation initiation factor eIF4GII). Genes with reduced translation are expected to have lower expression in RNA samples isolated from heavy polysomes but not in RNA samples isolated from whole cell lysates. LNSCs were grown in 10 cm plates, allowed to reach ≥ 80% confluency and then transfected with 400 pmol of control or Eif4g3 siRNA using Lipofectamine 2000. 7 plates were transfected with control or Eif4g3 siRNA. Total RNA was extracted from cells 48h after transfection. To isolate RNA from the heavy polysome fractions, cells were pooled, lysed and fractionated on a 10% to 60% continuous sucrose gradient. Fractions containing the heavy polysome fractions were pooled. RNA was extracted from these samples and used for microarrays on the Agilent Whole Mouse Genome Microarray Kit, 4×44K 2-color arrays.
Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 μg of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturer’s instructions. mRNA-seq samples were prepared from 1 μg of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 μg of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as ≥5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
Project description:Gametes rely heavily on post-transcriptional control mechanisms to regulate their differentiation. In eggs, the storage and selective temporal activation of maternal mRNAs is essential for normal development. In the male, transcription ceases during spermiogenesis necessitating the post-transcriptional regulation of many paternal mRNAs required for spermatid differentiation and spermatozoan function. Messenger RNAs that are being actively translated form polysomes. whereas translationally inactive mRNAs are often sequestered in ribonucleoproteins (RNPs). Here we combine polysome display and microarray analyses of RNP and polysome fractions of testes from prepuberal and adult mice to characterize the translation state of individual mRNAs as spermatogenesis proceeds.. Consistent with published reports, many post-meiotic mRNAs known to be translationally delayed shift from the RNPs into the polysomes, confirming the validity of this approach. In addition, based upon the criterion of movement from RNPs to polysomes, we detect another 742 mouse testicular genes showing dramatic shifts between RNPs and polysomes. One sub-group of 35 genes including the known translationally delayed Pgk2, are initially transcribed and translationally repressed in meiotic spermatocytes, and translated post-meiotically. This high-through-put approach defines the changing translation patterns of a large number of genes as male germ cells differentiate and identifies a new group of post-transcriptionally regulated meiotic transcripts for future study. Keywords: time course Overall design: Mouse testes from animals of 3 different ages were fractionated on a sucrose gradient and transcripts were quantified in the RNP and Polysome fractions. Transcripts were identified that changed their RNP/Polysome representation at the diffent developmental time points.
Project description:Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events that are driving these transitions. Here, we have used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation. Forty-eight hours (h) after PMSG injection, mice were stimulated with hCG for 0, 4, or 14 h, and GV-, MI- and MII-stage oocytes were collected. Polysome-bound mRNAs were purified, reverse-transcribed and linearly amplified with the WT-Ovation FFPE RNA Amplification System V2 (NuGEN). 5µg cDNA were fragmented and hybridized with Affymetrix Mouse Genome 430.2 array chips. Experiments were done using 3 independent sample sets.
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:Total RNA, low ribosome density (LRD) and polysome associated RNA were obtained from parallel S.coelicolor cultures of wild type strain MT1110 grown on solid medium SMMS up to rapid growth phase II. The abundance of each RNA in the total RNA, the LRD or polysome pool was quantified by Cydye labeling and hybridization on high density whole genome 105K S.coelicolor DNA microarrays using S.coelicolor M145 as reference.
Project description:Translational profiling of mouse cardiac tissue treated with 25mg/kg DMNQ in 10 ml/kg arachis oil over an acute time course (0.5-120 hours) compared to time matched control animals treated with 10ml/kg saline Two colour microarrays with time matched controls vs 25mg/kg DMNQ cardiac tissue. Before microarray analysis RNA separated on a sucrose density gradient into those mRNAs activitly undergoing translation (polysomes) and those not (monosomes) with control monosomes and treated monosomes on one set of arrays, and polysome control and polysome treated on another set of microarrays. The normalized Log2 of the monosomes was subtracted from the respective Log2 of the polysomes (on Series record). Time points studied were 0.5, 1, 2, 12, 24 and 120 hours following dosing, biological replicates n=3 independent animals at each time point, technical replicates (reverse labelling) n<1. One array printed onto two slides (A and B), one replicate per array.
Project description:Translational profiling of mouse cardiac tissue treated with 15mg/kg doxorubicin in 10 ml/kg saline over an acute time course (0.5-120 hours) compared to time matched control animals treated with 10ml/kg saline. Two colour microarrays with time matched controls vs 15mg/kg doxorubicin cardiac tissue. Before microarray analysis, RNA is separated on a sucrose density gradient into those mRNAs activity undergoing translation (polysomes) and those not (monosomes) with control monosomes and treated monosomes on one set of arrays, and polysome control and polysome treated on another set of microarrays. Thealized Log2 of the monosomes was subtracted from the respective Log2 of the polysomes (on Series record). Time points studied were 0.5, 1, 2, 12, 24 and 120 hours following dosing, biological replicates n=3 independent animals at each time point, technical replicates (reverse labelling) n<1. One array printed onto two slides (A and B), one replicate per array.
Project description:Heat stress is one of the most prominent and deleterious environmental threads affecting plant growth and development. Upon high temperatures, plants launch specialized gene expression programs that promote stress protection and survival. These programs involve global and specific changes at the transcriptional and translational levels. However the coordination of these processes and their specific role in the establishment of the heat stress response is not fully elucidated. In this report, we have carried out a genome-wide analysis to simultaneously monitor the individual changes in the transcriptional and translational mRNA levels of Arabidopsis thaliana seedlings after the exposure to a heat shock stress. Our results demonstrated that, superimposed to transcription, translation exerts a wide but dual regulation of gene expression. For the majority of mRNAs, translation is severely repressed, causing a decreased of 50% of the association of the bulk of mRNAs to polysomes. However, some relevant mRNAs involved in different aspects of homeostasis maintenance follow a differential pattern of translation. Analysis of the sequence of the differentially translated mRNAs unravels that some features, like the 5´UTR G+C content and the cDNA length, may take part in the discrimination mechanisms for mRNA polysome loading. Among the differential translated genes stand out master regulators of the stress response, highlighting the main role of translation in the early establishment of physiological response of plants to elevated temperatures. In total 8 ATH1 Affymetrix GeneChips were hybridized with all combinations of two factors: total mRNA/polysome-bound-RNA; 22ºC/38ºC. Two biological replicates per sample type were performed.
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. Overall design: rRNA-depleted RNA was extracted from pooled embryos of desired stages (Egg, 1-cell, 16-cells, 128-cells, 3.5hpf and 5.3hpf) and subjected to polysome profiling to separate between polysome-free and polysome-associated fragments. Unfractionated, rRNA-depleted samples at corresponding stages were used as background control. One RNA-seq library was generated for each sample.