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Global mRNA expression analysis in myo1 delta strains of the budding yeast Saccharomyces cerevisiae

ABSTRACT: The Saccharomyces cerevisiae MYO1 gene encodes the myosin type II heavy chain (Myo1p), a protein required for normal cytokinesis in budding yeast. Deletion of the MYO1 gene prevents actomyosin-driven cytokinesis thereby activating an alternative mechanism that involves the synthesis of a remedial septum. Myo1p deficiency in yeast (myo1) also causes the formation of attached cells, abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, and increased chitin synthesis. To determine how the differential expression of genes is related to these diverse phenotypes, we analyzed the global mRNA expression profile of myo1 strains. Global mRNA expression profiles of myo1 strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes were identified with 263 up-regulated and 284 down regulated genes in the myo1 strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. Genes involved in cell wall assembly (GAS1, PSA1, CIS3, FIT1, WSC2), MAP kinase activity (SLT2), Rho1p Guanine Exchange Factor (ROM1), and regulation of cell proliferation (RAS1) were also differentially expressed in myo1 strains. Conclusions: We have presented a global mRNA expression analysis of yeast myo1 strains and hypothesized about possible correlations with morphological and biochemical phenotypes observed in these strains. We report 547 differentially regulated genes in the myo1 mutant strains. Genes involved in the control of cell proliferation, protein synthesis and maturation, DNA replication, and cell division processes were largely down regulated, suggesting a mechanism for delayed cell cycle progression and growth that involves coordinated regulation of these processes in myo1 strains. Other genes involved in the cellular response to cell wall stress and cell wall organization were largely up-regulated suggesting that cell wall biogenesis is important for the completion of cytokinesis and cell wall morphogenetic processes that may also be affected by myosin II deficiency. Gene set enrichment analysis indicates that stress response and protein biosynthesis gene categories are inversely correlated in this mutant. Keywords: Comparative genomic hybridization

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE5931 | GEO | 2008/09/18

SECONDARY ACCESSION(S): PRJNA97381

REPOSITORIES: GEO

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Project description: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 (eIF2?-P) in myo1? strains and RPS8A, RPL7B and RPL3 mRNAs were found co-precipitated with immunoprecipitated eIF2?-P, suggesting a direct association between eIF2?-P and translationally regulated RP mRNAs. In yeast, GCN2 codes for the only eIF2? kinase that is directly regulated by TOR (target of rapamycin) pathway. Repression of TOR by rapamycin in a myo1? strain did not increase levels of eIF2?-P yet, a gcn2?myo1? 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 myo1? 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.

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Global mRNA expression analysis in myo1 delta strains of the budding yeast Saccharomyces cerevisiae

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