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Genetic screen of the yeast environmental stress response dynamics uncovers distinct regulatory phases.

ABSTRACT: Cells respond to environmental fluctuations by regulating multiple transcriptional programs. This response can be studied by measuring the effect of environmental changes on the transcriptome or the proteome of the cell at the end of the response. However, the dynamics of the response reflect the working of the regulatory mechanisms in action. Here, we utilized a fluorescent stress reporter gene to track the dynamics of protein production in yeast responding to environmental stress. The response is modulated by changes in both the duration and rate of transcription. We probed the underlying molecular pathways controlling these two dimensions using a library of ~1,600 single- and double-mutant strains. Dissection of the effects of these mutants and the interactions between them identified distinct modulators of response duration and response rate. Using a combination of mRNA-seq and live-cell microscopy, we uncover mechanisms by which Msn2/4, Mck1, Msn5, and the cAMP/PKA pathway modulate the response of a large module of stress-induced genes in two discrete regulatory phases. Our results and analysis show that transcriptional stress response is regulated by multiple mechanisms that overlap in time and cellular location.

SUBMITTER: Gutin J

PROVIDER: S-EPMC6711295 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Genetic screen of the yeast environmental stress response dynamics uncovers distinct regulatory phases.

Gutin Jenia J Joseph-Strauss Daphna D Sadeh Amit A Shalom Eli E Friedman Nir N

Molecular systems biology 20190801 8

Cells respond to environmental fluctuations by regulating multiple transcriptional programs. This response can be studied by measuring the effect of environmental changes on the transcriptome or the proteome of the cell at the end of the response. However, the dynamics of the response reflect the working of the regulatory mechanisms in action. Here, we utilized a fluorescent stress reporter gene to track the dynamics of protein production in yeast responding to environmental stress. The response ...[more]

PMID: 31464369

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Project description:BACKGROUND: Environmentally inflicted stresses such as salinity and drought limit the plant productivity both in natural and agricultural system. Increasing emphasis has been directed to molecular breeding strategies to enhance the intrinsic ability of plant to survive stress conditions. Functional screens in microorganisms with heterologous genes are a rapid, effective and powerful tool to identify stress tolerant genes in plants. Jatropha curcas (Physic nut) has been identified as a potential source of biodiesel plant. In order to improve its productivity under stress conditions to benefit commercial plantations, we initiated prospecting of novel genes expressed during stress in J. curcas that can be utilized to enhance stress tolerance ability of plant. RESULTS: To identify genes expressed during salt tolerance, cDNA expression libraries were constructed from salt-stressed roots of J. curcas, regulated under the control of the yeast GAL1 system. Using a replica based screening, twenty thousand yeast transformants were screened to identify transformants expressing heterologous gene sequences from J. curcas with enhanced ability to tolerate stress. From the screen we obtained 32 full length genes from J. curcas [GenBank accession numbers FJ489601-FJ489611, FJ619041-FJ619057 and FJ623457-FJ623460] that can confer abiotic stress tolerance. As a part of this screen, we optimized conditions for salt stress in J. curcas, defined parameters for salt stress in yeast, as well as isolated three salt hypersensitive yeast strains shs-2, shs-6 and shs-8 generated through a process of random mutagenesis, and exhibited growth retardation beyond 750 mM NaCl. Further, we demonstrated complementation of the salt sensitive phenotypes in the shs mutants, and analyzed the expression patterns for selected J. curcas genes obtained from the screen in both leaf and root tissues after salt stress treatments. CONCLUSIONS: The approach described in this report provides a rapid and universal assay system for large scale screening of genes for varied abiotic stress tolerance within a short span of time. Using this screening strategy we could isolate both genes with previously known function in stress tolerance as well as novel sequences with yet unknown function in salt stress tolerance from J. curcas. The isolated genes could be over-expressed using plant expression system to generate and evaluate transgenic plants for stress tolerance as well as be used as markers for breeding salt stress tolerance in plants.

Project description:By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G(1)/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G(1)-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G(1) arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.

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Genetic screen of the yeast environmental stress response dynamics uncovers distinct regulatory phases.

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Genetic screen of the yeast environmental stress response dynamics uncovers distinct regulatory phases.

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