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题目:: siRNA profiling of human HeLa cells response to UPF1 depletion
ID:
状态:: 发布时间June 15, 2008 , 更新时间 May 2, 2014 , 提交时间 Feb. 12, 2007,
物种:: Homo sapiens
摘要:: Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. Originally, NMD was thought of as a quality control pathway that targets non-functional mRNAs arising from mutations and splicing errors. More recently, NMD has been shown to also regulate normal gene expression and NMD thus emerged as one of the key post-transcriptional mechanisms of gene regulation. We have now systematically analyzed the molecular mechanism of variable NMD efficiency and used different HeLa cell strains as a model system. The results of this analysis show that NMD efficiency can be remarkably variable and represents a stable characteristic of these strains. Low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1 in one of the HeLa strain analyzed. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in the RNPS1 deficient cell line. We conclude that cellular concentrations of RNPS1 modify NMD efficiency and propose that the cell type specific co-factor availability represents a novel principle that controls NMD. Experiment Overall Design: HeLa cells were treated with UPF1 siRNA or Luciferase siRNA as a negative control. After 72 hs, cytoplasmic RNA was isolated and the integrity of the RNA was assessed using a Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). We performed preparation, processing, and hybridisation of labelled and fragmented cRNA targets to Affymetrix HG_U133A GeneChipsTM according to the manufacturer’s protocols (Affymetrix Inc., Santa Clara, CA). Oligonucleotide arrays were scanned using a confocal laser scanner (GeneArrayTM, Hewlett Packard, Palo Alto, CA). We used the Affymetrix GeneChip Suite 5.0 software (MAS 5.0) to calculate raw expression values for each of the 22,283 probe sets on the U133A oligonucleotide array. Signal intensities were calculated as average intensity difference (AID) between perfect and mismatch probes. Approximately 8,800 probe sets continuously resulting in absent calls were excluded from the analyses. Next, we used GeneSpring 4.2.1 (Silicon Genetics, Redwood City, CA) for scaling, normalisation and background correction of all genes and arrays. We performed Student´s t-test on normalised relative expression ratios to identify significant differentially expressed genes with a minimum factor of difference of >2-fold, within the 95% confidence interval (p<0.05).
实验种类:: RNAi profiling by array
样本量:: 6
实验设计:
: 无设计数据
数据号:: E-GEOD-7009, GSE7009
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