An Essential Role for the Saccharomyces cerevisiae DEAD-Box Helicase DHH1 in G1/S DNA-Damage Checkpoint Recovery

Bergkessel, Megan; Reese, Joseph C.

doi:10.1534/genetics.167.1.21

Cited by 30 publications

(31 citation statements)

References 52 publications

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“…1). Confirming prior results for other yeast genetic backgrounds (4,21), RCK/p54 and Xp54 also restored the capacity to grow at 35°C in the dhh1⌬ strain. Since expression of Xp54 and RCK/p54 achieved with 2m and the GPD promoter gave the highest level of TS Ϫ complementation, we chose these vectors for the experiments throughout.…”

supporting

confidence: 87%

Xenopus Xp54 and Human RCK/p54 Helicases Functionally Replace Yeast Dhh1p in Brome Mosaic Virus RNA Replication

Alves-Rodrigues

Más

Díez

2007

J Virol

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By using a Brome mosaic virus (BMV)-Saccharomyces cerevisiae system, we previously showed that the cellular Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex functions in BMV RNA translation and replication. As a first approach in investigating whether the corresponding human homologues play a similar role, we expressed human Lsm1p (hLsm1p) and RCK/p54 in yeast. Expression of RCK/p54 but not hLsm1p restored the defect in BMV RNA translation and replication observed in the dhh1⌬ and lsm1⌬ strains, respectively. This functional conservation, together with the common replication strategies of positive-stranded RNA viruses, suggests that RCK/p54 may also play a role in the replication of positive-stranded RNA viruses that infect humans.The group of positive-stranded RNA viruses includes major human pathogens such as hepatitis C virus and severe acute respiratory syndrome coronavirus. Since positive-stranded RNA viruses do not encapsidate the viral polymerase, upon entering the cell the viral RNA is first translated to produce viral replicases. Then, the viral RNA is recruited to membraneassociated replication complexes and used as a template for replication. This transition from translation to replication is a key step mediated by viral proteins as well as host factors (9, 15).The replication of Brome mosaic virus (BMV) in the yeast Saccharomyces cerevisiae is a well-established model system for studying common steps of positive-stranded RNA virus biology in a relatively simple genetic background (12). BMV is a plant virus with a tripartite positive-stranded RNA genome (1). RNA1 and RNA2 encode the replicases 1a, a helicase, and 2a, the polymerase. In the absence of 2a, 1a recruits the BMV positive-stranded RNA templates out of translation and into the endoplasmic reticulum, the site of replication. This recruitment dramatically increases the stability and accumulation of BMV positive-stranded RNAs (2,11,22). Finally, RNA3 encodes the movement and the capsid proteins. In this system, translation effects can easily be measured by following the translation efficiency of BMV RNA2 via Western blotting with 2a-specific antibodies (17), while recruitment of the viral RNA out of translation for replication can be studied via 1a-dependent RNA3 accumulation in Northern blots (12,15,19,20).With this BMV-yeast model system, we have recently shown that the yeast Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex, which functions in cellular mRNA degradation (6), is required for both translation and recruitment of BMV RNA to the replication complex (8,15,17). The Lsm1p-7p heptameric ring and the helicase Dhh1p belong to the highly conserved families of Sm/Sm-like proteins and DEAD-box helicase, respectively (13, 23). All the components of the Lsm1p-7p/Pat1p/ Dhh1p complex localize in cytoplasmic foci called P bodies. These are dynamic structures and sites of mRNA degradation, mRNA storage, and translation control (3, 6, 7). Interestingly, the Lsm1p-7p/Pat1p/Dhh1p complex mediates the movement of cellular mRNAs out of translation in...

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supporting

confidence: 87%

Xenopus Xp54 and Human RCK/p54 Helicases Functionally Replace Yeast Dhh1p in Brome Mosaic Virus RNA Replication

Alves-Rodrigues

Más

Díez

2007

J Virol

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“…(B) The ␤-galactosidase activities of plasmid FLO11-lacZ were measured in wild-type strain 10560-2B and mutant strains JK381 (caf20), JK384 (tif1), and JK387 (dhh1). ␤-Galactosidase assays were essentially the same as previously described (40 (4,8). Further analysis of the role of CAF20 and DHH1 in filamentous growth and STE12 expression, therefore, should help clarifying their roles in yeast cells.…”

Section: Discussionmentioning

confidence: 99%

Identification of Translational Regulation Target Genes during Filamentous Growth in Saccharomyces cerevisiae : Regulatory Role of Caf20 and Dhh1

Park

Hur

et al. 2006

Eukaryot Cell

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The dimorphic transition of yeast to the hyphal form is regulated by the mitogen-activated protein kinase and cyclic AMP-dependent protein kinase A pathways in Saccharomyces cerevisiae. Signaling pathway-responsive transcription factors such as Ste12, Tec1, and Flo8 are known to mediate filamentation-specific transcription. We were interested in investigating the translational regulation of specific mRNAs during the yeast-tohyphal-form transition. Using polyribosome fractionation and RT-PCR analysis, we identified STE12, GPA2, and CLN1 as translation regulation target genes during filamentous growth. The transcript levels for these genes did not change, but their mRNAs were preferentially associated with polyribosomes during the hyphal transition. The intracellular levels of Ste12, Gpa2, and Cln1 proteins increased under hyphal-growth conditions. The increase in Ste12 protein level was partially blocked by mutations in the CAF20 and DHH1 genes, which encode an eIF4E inhibitor and a decapping activator, respectively. In addition, the caf20 and dhh1 mutations resulted in defects in filamentous growth. The filamentation defects caused by caf20 and dhh1 mutations were suppressed by STE12 overexpression. These results suggest that Caf20 and Dhh1 control yeast filamentation by regulating STE12 translation.The cellular morphology of diploid Saccharomyces cerevisiae frequently switches between the yeast and filamentous forms depending on nutritional signals (16). Several signal transduction modules, including the mitogen-activated protein kinase (MAPK) cascade and the cyclic AMP-dependent protein kinase A (PKA) pathway, are known to participate in this switch (14,21,31,36). The MAPK cascade involves Ste20, Ste11, Ste7, Kss1, and the transcription factors Ste12 and Tec1 (15,21,29,30). The PKA pathway involves Gpr1, Gpa2, Ras2, Tpk2, and the transcription factors Flo8 and Sfl1 (22,26,31,32). These signaling pathways control the transcription of a number of filamentation-specific genes, including FLO11 (19,23,29).Although the signaling pathways and transcriptional regulation of yeast filamentous growth have been studied in considerable detail, little is known about translational regulation related to the transition from the yeast to the filamentous form. In this study, we searched for specific mRNAs that are preferentially translated during the yeast-to-hyphal-form transition. Genome-wide analysis of mRNA translation profiles indicates that the loading of ribosomes onto individual mRNA species varies broadly (20, 28). The association of mRNA transcripts in polyribosomes reflects the rate of synthesis of their corresponding proteins (3, 45). By purifying polyribosome fractions and employing RT-PCR analysis, we found that the mRNA transcripts of STE12, GPA2, and CLN1 were preferentially recruited to polyribosomes during filamentation compared to during normal vegetative growth, even though their levels in the total cell extracts were not changed. Consistently, the protein levels of Ste12, Gpa2, and Cln1 also increased during...

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“…P-bodies are cytoplasmic mRNA processing bodies (19), but it is unclear whether P-bodies degrade mRNA, serve as sites for mRNA storage, or both (20,21). P-bodies have been observed to form in response to a number of cellular stresses, primarily upon nutrient starvation, and have been indirectly implicated with the DNA damage response (22) and more recently in response to HU but not MMS (6). P-bodies were observed in Candida albicans upon various stresses, including UV irradiation (23).…”

Section: Significancementioning

confidence: 99%

A chemostat array enables the spatio-temporal analysis of the yeast proteome

Dénervaud

Becker

Delgado-Gonzalo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

124

151

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Observing cellular responses to perturbations is central to generating and testing hypotheses in biology. We developed a massively parallel microchemostat array capable of growing and observing 1,152 yeast-GFP strains on the single-cell level with 20 min time resolution. We measured protein abundance and localization changes in 4,085 GFP-tagged strains in response to methyl methanesulfonate and analyzed 576 GFP strains in five additional conditions for a total of more than 10,000 unique experiments, providing a systematic view of the yeast proteome in flux. We observed that processing bodies formed rapidly and synchronously in response to UV irradiation, and in conjunction with 506 deletion-GFP strains, identified four gene disruptions leading to abnormal ribonucleotide-diphosphate reductase (Rnr4) localization. Our microchemostat platform enables the large-scale interrogation of proteomes in flux and permits the concurrent observation of protein abundance, localization, cell size, and growth parameters on the single-cell level for thousands of microbial cultures in one experiment.O bserving proteins in the cellular milieu has been a longstanding technical challenge in biology. One major advance was the development of GFP, enabling the visualization of proteins in vivo (1). High-content imaging has been primarily applied to mammalian cells, using either reverse transfection arrays or microtiter-based systems in which the slow doubling time of mammalian cells enables long-term imaging under static conditions (2, 3).The Saccharomyces cerevisiae GFP fusion library covering 4,159 proteins provided the first static view of global protein abundance, localization, and noise (4, 5). This library was recently used to establish the static differences in protein abundance and localization in response to DNA replication stress induced by methyl methanesulfonate (MMS) and hydroxyurea (HU) (6), in response to DTT, H 2 O 2 , and nitrogen starvation (7), and 800 cytoplasmic proteins were analyzed upon entry into stationary phase (8). These three recent large-scale screens all relied on standard microtiter plates for imaging the yeast strains at a single time point before and after perturbation. Meanwhile, microfluidic devices emerged as powerful tools for conducting complex time-lapse experiments on small to medium scales (9, 10), enabling the analysis of cellular network responses (11) and the implementation of synthetically engineered systems (12). However, it has thus far been technically impossible to interrogate thousands of continuously growing microbial strains with high spatiotemporal resolution in a single experiment.Despite the fact that a wealth of systems-level information is available for S. cerevisiae, the single-cell temporal dynamics of protein abundance and localization has not yet been measured on a systems scale. To enable such analyses we developed a microfluidic platform capable of growing and observing 1,152 yeast strains with a temporal resolution of 20 min. We explored the dynamic behavior of ∼2/3 of the...

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An Essential Role for the Saccharomyces cerevisiae DEAD-Box Helicase DHH1 in G1/S DNA-Damage Checkpoint Recovery

Cited by 30 publications

References 52 publications

Xenopus Xp54 and Human RCK/p54 Helicases Functionally Replace Yeast Dhh1p in Brome Mosaic Virus RNA Replication

Xenopus Xp54 and Human RCK/p54 Helicases Functionally Replace Yeast Dhh1p in Brome Mosaic Virus RNA Replication

Identification of Translational Regulation Target Genes during Filamentous Growth in Saccharomyces cerevisiae : Regulatory Role of Caf20 and Dhh1

A chemostat array enables the spatio-temporal analysis of the yeast proteome

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