Membrane-association of mRNA decapping factors is independent of stress in budding yeast

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation

An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress

Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators (edc3∆ lsm4∆C), which result in defects in P body assembly, can destabilize mRNA under unstressed conditions. We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation

Nonsense-mediated mRNA decay controls the changes in yeast ribosomal protein pre-mRNAs levels upon osmotic stress

The expression of ribosomal protein (RP) genes requires a substantial part of cellular transcription, processing and translation resources. Thus, the RP expression must be tightly regulated in response to conditions that compromise cell survival. In Saccharomyces cerevisiae cells, regulation of the RP gene expression at the transcriptional, mature mRNA stability and translational levels during the response to osmotic stress has been reported. Reprogramming global protein synthesis upon osmotic shock includes the movement of ribosomes from RP transcripts to stress-induced mRNAs. Using tiling arrays, we show that osmotic stress yields a drop in the levels of RP pre-mRNAs in S. cerevisiae cells. An analysis of the tiling array data, together with transcription rates data, shows a poor correlation, indicating that the drop in the RP pre-mRNA levels is not merely a result of the lowered RP transcription rates. A kinetic study using quantitative RT-PCR confirmed the decrease in the levels of several RP-unspliced transcripts during the first 15 minutes of osmotic stress, which seems independent of MAP kinase Hog1. Moreover, we found that the mutations in the components of the nonsense-mediated mRNA decay (NMD), Upf1, Upf2, Upf3 or in exonuclease Xrn1, eliminate the osmotic stress-induced drop in RP pre-mRNAs. Altogether, our results indicate that the degradation of yeast RP unspliced transcripts by NMD increases during osmotic stress, and suggest that this might be another mechanism to control RP synthesis during the stress response

Checkpoints in a Yeast Differentiation Pathway Coordinate Signaling during Hyperosmotic Stress

All eukaryotes have the ability to detect and respond to environmental and hormonal signals. In many cases these signals evoke cellular changes that are incompatible and must therefore be orchestrated by the responding cell. In the yeast Saccharomyces cerevisiae, hyperosmotic stress and mating pheromones initiate signaling cascades that each terminate with a MAP kinase, Hog1 and Fus3, respectively. Despite sharing components, these pathways are initiated by distinct inputs and produce distinct cellular behaviors. To understand how these responses are coordinated, we monitored the pheromone response during hyperosmotic conditions. We show that hyperosmotic stress limits pheromone signaling in at least three ways. First, stress delays the expression of pheromone-induced genes. Second, stress promotes the phosphorylation of a protein kinase, Rck2, and thereby inhibits pheromone-induced protein translation. Third, stress promotes the phosphorylation of a shared pathway component, Ste50, and thereby dampens pheromone-induced MAPK activation. Whereas all three mechanisms are dependent on an increase in osmolarity, only the phosphorylation events require Hog1. These findings reveal how an environmental stress signal is able to postpone responsiveness to a competing differentiation signal, by acting on multiple pathway components, in a coordinated manner

The HOG Pathway Dictates the Short-Term Translational Response after Hyperosmotic Shock

In the global osmoshock translational response in yeast, some gene products were translationally mobilized without transcriptional up-regulation. Conversely, other transcriptionally up-regulated mRNAs were translationally inhibited. Analogous changes occurred on the protein level. These translational responses were strongly dependent on Hog1 and Rck2

Pag1p, a Novel Protein Associated with Protein Kinase Cbk1p, Is Required for Cell Morphogenesis and Proliferation in Saccharomyces cerevisiae

Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae, has previously been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1, a novel gene in S. cerevisiae, displayed defects similar to those of cbk1 mutants. pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 and CBK1 act in the same pathway. Furthermore, we found that Pag1p and Cbk1p localize to the same polarized peripheral sites and that they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes