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GSE19719
Homo sapiens
2 Downloadable Samples
Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3UTRs, 5UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, our data suggest that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.
Publication Title
Alu sequences in undifferentiated human embryonic stem cells display high levels of A-to-I RNA editing.
Sample Metadata Fields
Specimen part
View Samples- Escherichia coli str. k-12 substr. mg1655
- 89 Downloadable Samples
- Affymetrix E. coli Genome 2.0 Array (ecoli2)
Description
Our laboratory has recently discovered that E. coli cells starved for the DNA precursor dGTP are killed efficiently (dGTP starvation) in a manner similar to that described for Thymineless Death (TLD). Conditions for specific dGTP starvation can be achieved by depriving an E. coli optA1 gpt strain of the purine nucleotide precursor hypoxanthine (Hx). To gain insight into the mechanisms underlying dGTP starvation, we conducted genome-wide gene expression analyses on actively growing optA1 gpt strains subjected to hypoxanthine deprivation for increasing periods of time. The data show that, upon Hx withdrawal, the optA1 gpt strain displays a diminished ability to de-repress the de novo purine biosynthesis genes, and this is likely due to internal guanine accumulation. The impairment to fully induce the purR regulon may be a contributing factor to the lethality of dGTP starvation. At later time points, and coinciding with cell lethality, strong induction of the SOS is observed, supporting the concept of replication stress as a final cause of death. No evidence was observed for the participation of other stress responses, including the rpoS-mediated global stress response in the starved cells, and reinforcing the lack of feedback of replication stress into the global metabolism of the cell. The genome-wide expression data also provide direct evidence for increased genome complexity during dGTP starvation, as a markedly increased gradient is observed for expression of genes located nearby the replication origin relative to those located towards the replication terminus.
Publication Title
Transcriptome Analysis of Escherichia coli during dGTP Starvation.
Sample Metadata Fields
No sample metadata fields
View Samples- Drosophila melanogaster
- 12 Downloadable Samples
Illumina HiSeq 2000
Description
Purpose: Investigating the role of Drosophila G9a in oxidative stress responses. Methods: Flies were collected after eclosion and allowed to recover from CO2 exposure for 5 days prior to paraquat exposure. Paraquat (Methyl viologen dichloride hydrate 98 %; Sigma 856177) was mixed into the flyfood at 40 °C to a final concentration of 50 mM. For OS induction, 5-9 day old flies were transferred to paraquat containing food and incubated at 25 °C and 70 % humidity. At each time point, flies were flash frozen in liquid nitrogen followed by vortexing and filtering through a series of sieves to isolate heads from other body parts. 200 fly heads per sample were used for RNA extraction using QIAGEN lipid mini tissue kit. The TruSeq RNA Sample Preparation Kit v2 (Illumina) was used to prepare adapter ligated PCR fragments for sequencing. PCR was used to selectively enrich the fragments containing the adapters. The PCR fragments were validated using Agilent 2200 TapeStation. Single indexed samples were multiplexed and sequenced on an Illumina HiSeq 2000 sequencing system (Illumina) in single-end mode with a read length of 35 bp. Quality of sequenced reads was assessed with FastQC. The RNAseq experiments were conducted on two biological duplicates for each condition. Sequenced reads were aligned with Burrows-Wheeler algorithm (BWA) (Li & Durbin, 2010) to the Drosophila reference genome (BDGP.5, http://www.fruitfly.org/) and per gene read counts were generated with HTSeq count (Anders et al, 2015). 25–30 million reads with high quality alignment were obtained for each sample and used for differential expression analysis. DESeq (Anders & Huber, 2010) was used to obtain library size-normalized read counts and to calculate differential expression of genes in 4 pairwise comparisons: 0 h versus 6 h and 0 h versus 12 h after OS in both G9a mutants and controls (fold change =1.5, adjusted p-value= 0.05, Benjamini-Hochberg). Results: We found 2731 genes to be differential expressed in at least one of the four pairwise comparisons. The largest group of differentially expressed (DE) genes are highly augmented upon OS induction in the G9a mutant (41.7 % of all DE genes). The second largest group of DE genes (23.9 % of all DE genes) were more downregulated in G9a mutant in response to OS. Genes that are over-activated in G9a mutants are predominantly involved in OS response and OS mediated damage, whereas genes that are downregulated in G9a mutants are involved in energy metabolism. Conclusions: Our data suggest that G9a provides an epigenetic mechanism that safeguards an appropriate transcriptional response to OS and preserves immediately available energy, thereby acting as a critical regulatory hub between the transcriptional and physiological responses to oxidative stress. Overall design: fly-head mRNA libraries of 5-9 days old male G9aDD1 mutant and control during 0, 6 and 12 hours of paraquat oxidative stress exposure were sequenced in duplicate on Hi-seq 2000.
Publication Title
The histone methyltransferase G9a regulates tolerance to oxidative stress-induced energy consumption.
Sample Metadata Fields
Specimen part, Treatment, Subject
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