TSAA
Ontology highlight
ABSTRACT:
ORGANISM(S): Aspergillus fumigatus
SUBMITTER: David Askew
PROVIDER: E-MTAB-2027 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The unfolded protein response (UPR) is a network of intracellular signaling pathways that supports the ability of the secretory pathway to maintain equilibrium between the load of proteins entering the endoplasmic reticulum (ER) and the protein folding capacity of the ER lumen. Current evidence suggests that human pathogenic fungi rely heavily on this pathway for virulence, but there is limited understanding of the mechanisms involved. The best known functional output of the UPR is transcriptional upregulation of mRNAs involved in ER homeostasis. However, this does not take into account mechanisms of translational regulation that involve differential recruitment of mRNAs to ribosomes. In this study, a global analysis of transcript-specific translational regulation was performed in the pathogenic mold Aspergillus fumigatus to determine the nature and scope of the translational response to ER stress.
2014-01-13 | E-MTAB-2028 | biostudies-arrayexpress
Project description:CRZ is a C2H2 zinc finger transcription factor activated by calcineurin, a protein phosphatase activated by calcium-calmodulin, in different stress conditions related with calcium cell accumulation. CRZ binds to a specific element on the promoter region of genes called CDRE (calcineurin-dependent response element) shown to be sufficient for calcium- and calcineurin-dependent gene expression. In order to investigate which pathways are involved under calcium stress conditions, we determined the transcriptional profile of A. fumigatus delta crzA mutant strain upon a time course 200 mM calcium chloride treatment. Conidia from the mutant and wild type strains were incubated at 37°C in complete medium for 16 hours and were challenged with 200 mM calcium chloride for 10 and 30 minutes. At each time point, the mycelia were harvested by centrifugation and used for competitive microarray hybridizations. We were able to observe a decreased mRNA expression of genes encoding inorganic ion transport proteins and lipid transport. In contrast, increased mRNA expression was observed for several genes involved in transcription, energy production and conversion, cell cycle control, cell division, chromosome partitioning, amino acid, lipid and carbohydrate transport and metabolism, nucleotide transport, cell wall and membrane biogenesis, posttranslation modifications, protein turnover, chaperones, inorganic ion transport, signal transduction mechanisms, intracellular trafficking, secretion and vesicular transport. Keywords: gene expression array-based (log2 ratio) For the time course microarray experiments, 5.0 x 108 conidia of A. fumigatus wild type [CEA17 delta akuB(KU80)] and delta crzA strains were inoculated in 300 ml of pre-warmed liquid cultures (YG) in 500-mL erlenmeyer flasks and allowed to grow for 16 hours in a reciprocal shaker (250 rpm) at 37°C. After this time, the cultures were added by calcium chloride (200 mM) and incubated for 10 and 30 more minutes before mycelia recovery and RNA extraction. Hybridization experiments were competitive using probes derived from calcium chloride shock cultures using the wild type strain exposed to calcium (10 or 30 minutes) as the reference RNA for every hybridizantion. Normalized signal intensities were used to generate relative hybridization ratios (query/reference). Following normalization, the values for each gene's in-slide replicates were averaged (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects. These final, processed data are linked below as supplementary files.
2010-06-20 | E-GEOD-15432 | biostudies-arrayexpress
Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Perk wild-type or knockout mouse liver were perfused without or with 2,4-di-tert-butylhydroquinone (tBuHQ) for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Perk+/+, -tBuHQ or +tBuHQ; total and polysome fraction of Perk-/-, -tBuHQ or +tBuHQ. Each array analysis was done in duplicate.
2009-11-23 | E-GEOD-11684 | biostudies-arrayexpress
Project description:The initial translational response of the yeast Saccharomyces cerevisiae in response to acetic acid-induced apoptosis was investigated by microarray profiling of mRNAs contained in polysomal fractions obtained upon 0 (used as control), 15 and 30 minutes of acetic acid treatment. The mRNA fraction thus investigated corresponds to the mRNAs capable of overcoming the inhibition of cap-mediated translation initiation.
2013-02-24 | E-MEXP-3570 | biostudies-arrayexpress
Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. This SuperSeries is composed of the following subset Series:; GSE11496: Expression data from Gcn2 wild-type and knockout mouse liver perfused with or without methionine; GSE11684: Expression data from Perk wild-type and knockout mouse liver perfused without or with 2,5-di-tert-butylhydroquinone Experiment Overall Design: Refer to individual Series
2009-11-23 | E-GEOD-11685 | biostudies-arrayexpress
Project description:The purpose of the experiment is to identify mRNAs under differential translational control in Ki-ras transformed 267B1 cells as compared to the parental cell line. Total and polysome-associated mRNAs were isolated from exponentially growing cells and used to prepare biotin-labeled cRNA probes for analysis on Affymetrix microarray chips as described previously. The Data Mining tool (Affymetrix) was used to identify mRNAs that were differentially increased or decreased only in polysome fractions.
2007-06-26 | E-MEXP-461 | biostudies-arrayexpress
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 M-NM-<g 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 manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g 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 M-NM-<g 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 M-bM-^IM-%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).
2014-06-12 | E-GEOD-52799 | biostudies-arrayexpress
Project description:Transcription factor Msn2 played crucial roles in mediating fungal stress tolerance, a determinant to the biocontrol potential of fungal entomopathogens. We characterized for the first time the functions of Beauveria bassiana Msn2 (BbMsn2) and Metarhizium robertsii (MrMsn2) by analyzing multi-phenotypic changes in Msn2-deletion and investigating transcription patterns of WT versus M-NM-^TMsn2 of B. bassiana and M. robertsii under thermal and oxidative stresses by using high throughput sequencing (RNA-Seq). Our transcriptional profiles revealed that numerous differentially expressed genes (DEGs), of which involved in transportation, detoxification, signal transduction, and energy metabolism, were significantly repressed in expression level. Total RNA obtained from Bbmsn2 and MrMsn2 disruption mutant subjected to 2 mM menadione and 40 M-BM-0C for 3-h response compared to the wild type strain under the same stress treatment.
2015-02-01 | E-GEOD-40765 | biostudies-arrayexpress
Project description:To gain a panoramic view of Smaug function in the early embryo we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified the mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug?s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets as well as many metabolic enzymes, including several glycolytic enzymes. Embryos laid by wild-type or smaug1 homozygous mothers were collected 0-2 hours post-egglaying and lysed in an equal volume of polysome lysis buffer. The lysate was applied to polysome gradients that were fractionated into four fractions. mRNA was extracted from each fraction and converted to cDNA that was applied on custom-designed Nimblegen arrays. Three biological replicates were performed for each of the four fraction in each of the two genotypes. To demonstrate that the presence of mRNAs at the bottom of the gradient were the result of translation, we performed a puromycin experiment. smaug1 homozygous mothers were collected 0-2 hours post-egglaying and lysed in an equal volume of puromycin lysis buffer and then treated either with cycloheximide or puromycin. The lysate from each condition was applied to separate polysome gradients that were each fractionated into four fractions. mRNA was extracted from each fraction and converted to cDNA that was applied on custom-designed Nimblegen arrays. Two biological replicates were performed for each of the four fraction from each of the two conditions.
2013-12-03 | E-GEOD-50026 | biostudies-arrayexpress
Project description:Malaria is caused by Plasmodium parasites, which are transmitted via the bites of infected Anopheline mosquitoes. Midgut invasion is a major bottleneck for Plasmodium development inside the mosquito vectors as a rapidly responding immune system recognizes ookinetes and recruits killing factors from the midgut and surrounding tissues, dramatically reducing the population of invading ookinetes before they can successfully traverse the midgut epithelium. Understanding molecular details of the parasite-vector interactions requires precise measurement of nascent protein synthesis in the mosquito during Plasmodium infection. Current expression profiling primarily monitors alterations in steady-state levels of mRNA, but does not address the equally critical issue of whether the proteins encoded by the mRNAs are actually synthesized. In this study, we used sucrose density gradient centrifugation to isolate actively translating Anopheles gambiae mRNAs based upon their association with polyribosomes (polysomes). The proportion of individual gene transcripts associated with polysomes, which is determined by RNA deep sequencing, reflects mRNA translational status. This approach led to identification of 1017 mosquito transcripts that were primarily regulated at the translational level after ingestion of Plasmodium falciparum-infected blood. Caspar, a negative regulator of the NF-kappaB transcription factor Rel2, appears to be substantially activated at the translational levels during Plasmodium infection. In addition, transcripts of Dcr1, Dcr2 and Drosha, which are involved in small RNA biosynthesis, exhibited enhanced associations with polysomes after P. falciparum challenge. This observation suggests that mosquito microRNAs may play an important role in reactions against Plasmodium invasion. We analyzed both total cellular mRNAs and mRNAs that are associated with polysomes to simultaneously monitor transcriptomes and nascent protein synthesis in the mosquito. This approach provides more accurate information regarding the rate of protein synthesis, and identifies some mosquito factors that might have gone unrecognized because expression of these proteins is regulated mainly at the translational level rather than at the transcriptional level after mosquitoes ingest a Plasmodium-infected blood meal. Midguts from female Anopheles gambiae mosquitoes were dissected at around 24 h after ingestion of P. falciparum-infected blood. Mosquitoes fed on uninfected blood were used as control. A portion of the midguts were used for isolation of total cellular RNA. Extracts of the remaining midguts were fractionated over sucrose density gradients, and fifteen fractions were collected from the top of each gradient. Non-polysomal fractions, as well as polysomal fractions were combined, respectively, to obtain two RNA pools per gradient for three independent experiments. The first, last and the sample between non-polysomal and polysomal, were all discarded to ensure pure pools from each set. A fourth experiment was conducted where the polysomal and non-polysomal samples were not pooled, to be used later for qRT-PCR. The mRNA levels of individual mosquito genes in polysome (PS) fractions, nonpolysome (NP) fractions and unfractionated steady-state (total) RNA were determined using high throughput RNA deep sequencing. Each RNA pool generated 2.1-9.8 million raw read . Signal intensities of transcripts from each PS pool were compared to those of transcripts from a matching NP pool. To quantify the translational status of individual mRNA species, we define the relative PS loading [PL=P/(P+NP)] as the extent of mRNA association with polysomes. PL values were compared between the mosquitoes fed on P. falciparum-infected or -uninfected blood.
2012-09-21 | E-GEOD-38707 | biostudies-arrayexpress