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题目:: Post-transcriptional regulation of stress response in a myo1Δ mutant of the budding yeast Saccharomyces cerevisiae
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状态:: 发布时间Dec. 8, 2010 , 更新时间 May 1, 2014
物种:: Saccharomyces cerevisiae
摘要:: The microarrays experiments of three biological and one technical replicates were performed in YJR12 (wt) and YJR13 (myo1∆) strains. YJR12 (wild type) and YJR13 (myo1∆) strains were obtained as haploid segregants from a cross between YJR6 (myo1::HIS5 strain) and BY4741 (obtained from ATTC). Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base) with continuous shaking at 200 rpm. RNA was extracted from ribosomal pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies). The purity and integrity of the RNA was monitored using an Agilent Bioanalyzer (Agilent Technologies) following the manufacturer’s instructions. 1.0 µg of RNA extracted form ribosomal pellets from each sample was amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies). The amplified cRNA was labeled with 10mM Cyanine 5-CTP (Cy5) or Cyanine 3-CTP (Cy3) (Perkin Elmer Life Sciences). Labeled cRNA’s were purified with Qiagen RNeasy mini spin columns and dye incorporation was monitored on an Agilent Bioanalyzer. Hybridization of Cy5 and Cy3 labeled cRNA’s were performed using Yeast Oligo Microarray slides and hybridization kit from Agilent Technologies (Sheldon Manufacturing) at 60ºC for 17 hours. Slides were washed and scanned with a VersArray Chip Reader system (BioRad, Hercules, CA) at a resolution of 5mm with detector sensitivity values between 704-800 and laser power at 85%. Scanned images were transferred to the Imagene 3.0 software (Biodiscovery) for further analysis to locate spots, adjust the appropriate grid, and obtain the Cy3 and Cy5 TIFF files. The microarrays raw data generated with Imagene 3.0 were analyzed using Limma software (Bioconductor Package 1.7). The data was prepared for analysis by correcting for background intensity. The individual data sets were normalized using the locally weighted linear regression (Lowess) within each array. After normalization, the difference between the experimental and control signal was calculated, replicates were combined, and their averages were calculated. The fold change in gene expression was calculated by 2^(M), where M is the log2-fold change after background correction and normalization. An Empirical Bayes Statistics for differential expression analysis (eBayes statistics) was performed by Limma. Genes with a p-value ≤ 0.018 were established as a cutoff for differential expression. In addition, a false discovery rate (FDR) test was performed by Limma program. Previous analysis of global mRNA expression in Saccharomyces cerevisiae myosin type II deficient strains (myo1∆) revealed 547 genes related to the stress response that were proposed to be regulated at the transcriptional level. The objective of this study is to explore the post-transcriptional regulation of the stress response in these strains. We have identified 1,271 differentially regulated mRNAs bound to ribosomes extracted from myo1∆ strains compared to wild type controls. To assess the mode of regulation of these putative translationally regulated genes, ribosomal protein (RP) mRNAs were analyzed for their polysomal distribution in sucrose gradient fractions of myo1∆ and wild-type (wt) cells. Our analysis of three representative RP mRNAs showed that RPS8A, RPL7B and RPL3 were recruited from heavy to lighter polyribosome fractions in myo1∆, suggesting that these RP mRNAs were less efficiently translated. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2α-P) in myo1Δ strains and RPS8A, RPL7B and RPL3 mRNAs were found co-precipitated with immunoprecipitated eIF2α-P, suggesting a direct association between eIF2α-P and translationally regulated RP mRNAs. In yeast, GCN2 codes for the only eIF2α kinase that is directly regulated by TOR (target of rapamycin) pathway. Repression of TOR by rapamycin in a myo1Δ strain did not increase levels of eIF2α-P yet, a gcn2Δmyo1Δ strain exhibited a severe synthetic growth defect suggesting an important survival role for TOR mediated translational regulation in these strains. Reduced steady state levels of the translation initiation factor eIF4G, were also observed in myo1Δ further supporting the regulation of translation by the TOR pathway. These findings support the conclusion that post-transcriptional control specifically by translation inhibition is a key component of the stress response in yeast.
实验种类:: transcription profiling by array
样本量:: 8
实验设计:
: 无设计数据
数据号:: E-GEOD-20203, GSE20203
数据状态:: 无法自动分析，您可以尝试手动分析数据。

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