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Apparent ploidy effects on silencing are post-transcriptional at HML and telomeres in Saccharomyces cerevisiae.

ABSTRACT: The repression of genes in regions of heterochromatin is known as transcriptional silencing. It occurs in a wide range of organisms and can have importance in adaptation to the environment, developmental changes and disease. The model organism Saccharomyces cerevisiae has been used for many years to study transcriptional silencing, but until recently no study has been made in relation to ploidy. The aim of this work was to compare transcriptional silencing in haploids and diploids at both telomeres and the hidden mating-type (HM) loci. Transcriptional silencing was assayed, by growth on 5-fluoroorotic acid (5-FOA) media or by flow cytometry, on strains where a telomere or HM locus was marked. RNA levels were measured by quantitative RT-PCR to confirm that effects were transcriptional. 5-FOA assays and flow cytometry were consistent with transcriptional silencing at telomeres and at HML being reduced as ploidy increases which agreed with conclusions in previous publications. However, QRT-PCR revealed that transcriptional silencing was unaffected by ploidy and thus protein levels were increasing independently of RNA levels. At telomere XI left (XI-L), changes in protein level were strongly influenced by mating-type, whereas at HML mating-type had much less influence. The post-transcriptional effects seen in this study, illustrate the often ignored need to measure RNA levels when assaying transcriptional silencing in Saccharomyces cerevisiae.

SUBMITTER: McLaughlan JM

PROVIDER: S-EPMC3392252 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Apparent ploidy effects on silencing are post-transcriptional at HML and telomeres in Saccharomyces cerevisiae.

McLaughlan Jenny M JM Liti Gianni G Sharp Sarah S Maslowska Agnieszka A Louis Edward J EJ

PloS one 20120709 7

The repression of genes in regions of heterochromatin is known as transcriptional silencing. It occurs in a wide range of organisms and can have importance in adaptation to the environment, developmental changes and disease. The model organism Saccharomyces cerevisiae has been used for many years to study transcriptional silencing, but until recently no study has been made in relation to ploidy. The aim of this work was to compare transcriptional silencing in haploids and diploids at both telome ...[more]

PMID: 22792162

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Project description:Saccharomyces cerevisiae myosin type II-deficient (myo1?) strains remain viable and divide, despite the absence of a cytokinetic ring, by activation of the PKC1-dependent cell wall integrity pathway (CWIP). Since the myo1? transcriptional fingerprint is a subset of the CWIP fingerprint, the myo1? strain may provide a simplified paradigm for cell wall stress survival.To explore the post-transcriptional regulation of the myo1? stress response, 1,301 differentially regulated ribosome-bound mRNAs were identified by microarray analysis of which 204 were co-regulated by transcription and translation. Four categories of mRNA were significantly affected - protein biosynthesis, metabolism, carbohydrate metabolism, and unknown functions. Nine genes of the 20 CWIP fingerprint genes were post-transcriptionally regulated. Down and up regulation of selected ribosomal protein and cell wall biosynthesis mRNAs was validated by their distribution in polysomes from wild type and myo1? strains. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2?-P) and a reduction in the steady state levels of the translation initiation factor eIF4Gp in myo1? strains. Deletion of GCN2 in myo1? abolished eIF2?p phosphorylation, and showed a severe growth defect. The presence of P-bodies in myo1? strains suggests that the process of mRNA sequestration is active, however, the three representative down regulated RP mRNAs, RPS8A, RPL3 and RPL7B were present at equivalent levels in Dcp2p-mCh-positive immunoprecipitated fractions from myo1? and wild type cells. These same RP mRNAs were also selectively co-precipitated with eIF2?-P in myo1? strains.Quantitative analysis of ribosome-associated mRNAs and their polyribosome distributions suggests selective regulation of mRNA translation efficiency in myo1? strains. Inhibition of translation initiation factor eIF2? (eIF2?-P) in these strains was by Gcn2p-dependent phosphorylation. The increase in the levels of eIF2?-P; the genetic interaction between GCN2 and MYO1; and the reduced levels of eIF4Gp suggest that other signaling pathways, in addition to the CWIP, may be important for myo1? strain survival. Selective co-immunoprecipitation of RP mRNAs with eIF2?-P in myo1? strains suggests a novel mode of translational regulation. These results indicate that post-transcriptional control is important in the myo1? stress response and possibly other stresses in yeast.

Project description:BackgroundGross chromosomal rearrangements (GCRs) such as aneuploidy are key factors in genome evolution as well as being common features of human cancer. Their role in tumour initiation and progression has not yet been completely elucidated and the effects of additional chromosomes in cancer cells are still unknown. Most previous studies in which Saccharomyces cerevisiae has been used as a model for cancer cells have been carried out in the haploid context. To obtain new insights on the role of ploidy, the cellular effects of GCRs were compared between the haploid and diploid contexts.ResultsA total number of 21 haploid and diploid S. cerevisiae strains carrying various types of GCRs (aneuploidies, nonreciprocal translocations, segmental duplications and deletions) were studied with a view to determining the effects of ploidy on the cellular responses. Differences in colony and cell morphology as well as in the growth rates were observed between mutant and parental strains. These results suggest that cells are impaired physiologically in both contexts. We also investigated the variation in genomic expression in all the mutants. We observed that gene expression was significantly altered. The data obtained here clearly show that genes involved in energy metabolism, especially in the tricarboxylic acid cycle, are up-regulated in all these mutants. However, the genes involved in the composition of the ribosome or in RNA processing are down-regulated in diploids but up-regulated in haploids. Over-expression of genes involved in the regulation of the proteasome was found to occur only in haploid mutants.ConclusionThe present comparisons between the cellular responses of strains carrying GCRs in different ploidy contexts bring to light two main findings. First, GCRs induce a general stress response in all studied mutants, regardless of their ploidy. Secondly, the ploidy context plays a crucial role in maintaining the stoichiometric balance of the proteins: the translation rates decrease in diploid strains, whereas the excess protein synthesized is degraded in haploids by proteasome activity.

Dataset Information

Apparent ploidy effects on silencing are post-transcriptional at HML and telomeres in Saccharomyces cerevisiae.

Publications

Apparent ploidy effects on silencing are post-transcriptional at HML and telomeres in Saccharomyces cerevisiae.

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