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Series GSE5324 Query DataSets for GSE5324
Status Public on Oct 17, 2006
Title TTP mRNA targets identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts
Organism Mus musculus
Experiment type Expression profiling by array
Summary Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice, and cells derived from them, showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target
mRNAs for TTP include tumor necrosis factor alpha (TNF), granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 2 beta (IL2 beta). Here we used microarray analysis of RNA from wild-type and TTP-deficient fibroblast cell lines to identify transcripts with different decay rates, after serum stimulation and actinomycin D treatment. Of 250 mRNAs apparently stabilized in the absence of TTP, 23 contained conserved TTP binding sites; 10 of these were shown by secondary analyses to be stabilized. The most dramatically affected transcript encoded the protein Ier3, recently implicated in the physiological control of blood pressure. The Ier3 transcript contained several conserved TTP binding sites that could bind TTP directly, and conferred TTP sensitivity to the mRNA in cell transfection studies. These studies have identified several new, physiologically relevant TTP target transcripts in fibroblasts; these target mRNAs encode proteins from a variety of functional classes.
Keywords: mRNA degradation, time course, knockout experiment
 
Overall design Cell lines were derived from mouse embryonic fibroblast (MEF) cultures from littermate E14.5 embryos (for Zfp36 +/+ and -/- cells). MEFs were maintained at 37o C (5% CO2) in DMEM containing 10% FBS, 100
U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. When the cells had reached approximately 70-90 % confluence, they were trypsinized and diluted 2-50 fold in the same medium, based on morphology and growth rate, such that they would reach near confluence in 3-4 days when subcultured.

Five identical experiments involving sequential serum deprivation, serum stimulation and actinomycin D treatment were performed on five different days; each experiment involved simultaneous identical experiments with the WT and KO cell lines. In each experiment, the cells were serum deprived for 16h, and a sample was taken at that point as a serum-deprived control. The cells were then stimulated by 10% FBS for
90 min and another sample taken, after which 5μg/ml actinomycin D was added; samples for RNA analysis were then removed at 30, 60, 90 and 120 min after actinomycin D treatment. In all experiments, each sample represented three combined 100 mm dishes of cells.
 
Contributor(s) Lai WS, Parker JS, Grissom SF, Stumpo DJ, Blackshear PJ
Citation(s) 17030620
Submission date Jul 17, 2006
Last update date Feb 11, 2019
Contact name NIEHS Microarray Core
E-mail microarray@niehs.nih.gov, liuliw@niehs.nih.gov
Organization name NIEHS
Department DIR
Lab Microarray Core
Street address 111 T.W. Alexander Drive
City RTP
State/province NC
ZIP/Postal code 27709
Country USA
 
Platforms (1)
GPL1261 [Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array
Samples (48)
GSM120229 TTP_KO_0ActD_BioRep1
GSM120230 TTP_KO_30ActD_BioRep1
GSM120231 TTP_KO_60ActD_BioRep1
Relations
BioProject PRJNA96335

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE5324_RAW.tar 290.7 Mb (http)(custom) TAR (of CEL)
Raw data provided as supplementary file

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