Paula Alepuz

Specific and global regulation of mRNA stability during osmotic stress in Saccharomyces cerevisiae

Hyperosmotic stress yields reprogramming of gene expression in Saccharomyces cerevisiae cells. Most of this response is orchestrated by Hog1, a stress-activated, mitogen-activated protein kinase (MAPK) homologous to human p38. We investigated, on a genomic scale, the contribution of changes in transcription rates and mRNA stabilities to the modulation of mRNA amounts during the response to osmotic stress in wild-type and hog1 mutant cells. Mild osmotic shock induces a broad mRNA destabilization; however, osmo-mRNAs are up-regulated by increasing both transcription rates and mRNA half-lives. In contrast, mild or severe osmotic stress in hog1 mutants, or severe osmotic stress in wild-type cells, yields global mRNA stabilization and sequestration of mRNAs into P-bodies. After adaptation, the absence of Hog1 affects the kinetics of P-bodies disassembly and the return of mRNAs to translation. Our results indicate that regulation of mRNA turnover contributes to coordinate gene expression upon osmotic stress, and that there are both specific and global controls of mRNA stability depending on the strength of the osmotic stress.

Eukaryotic mRNA Decay: Methodologies, Pathways, and Links to Other Stages of Gene Expression

mRNA concentration depends on the balance between transcription and degradation rates. On both sides of the equilibrium, synthesis and degradation show, however, interesting differences that have conditioned the evolution of gene regulatory mechanisms. Here, we discuss recent genome-wide methods for determining mRNA half-lives in eukaryotes. We also review pre- and posttranscriptional regulons that coordinate the fate of functionally related mRNAs by using protein- or RNA-based trans factors. Some of these factors can regulate both transcription and decay rates, thereby maintaining proper mRNA homeostasis during eukaryotic cell life.

A Gene-Specific Requirement for FACT during Transcription Is Related to the Chromatin Organization of the Transcribed Region

ABSTRACT The FACT complex stimulates transcription elongation on nucleosomal templates. In vivo experiments also involve FACT in the reassembly of nucleosomes traversed by RNA polymerase II. Since several features of chromatin organization vary throughout the genome, we wondered whether FACT is equally required for all genes. We show in this study that the in vivo depletion of Spt16, one of the subunits of Saccharomyces cerevisiae FACT, strongly affects transcription of three genes, GAL1, PHO5, and Kluyveromyces lactis LAC4, which exhibit positioned nucleosomes at their transcribed regions. In contrast, showing a random nucleosome structure, YAT1 and Escherichia coli lacZ are only mildly influenced by Spt16 depletion. We also show that the effect of Spt16 depletion on GAL1 expression is suppressed by a histone mutation and that the insertion of a GAL1 fragment, which allows the positioning of two nucleosomes, at the 5′ end of YAT1 makes the resulting transcription unit sensitive to Spt16 depletion. These results indicate that FACT requirement for transcription depends on the chromatin organization of the 5′ end of the transcribed region.

Yeast mRNA cap-binding protein Cbc1/Sto1 is necessary for the rapid reprogramming of translation after hyperosmotic shock

Global translation is inhibited in Saccharomyces cerevisiae cells under osmotic stress; nonetheless, osmostress-protective proteins are synthesized. We found that translation mediated by the mRNA cap-binding protein Cbc1 is stress-resistant and necessary for the rapid translation of osmostress-protective proteins under osmotic stress.

eIF5A facilitates translation termination globally and promotes the elongation of many non polyproline-specific tripeptide sequences.

Abstract eIF5A is an essential protein involved in protein synthesis, cell proliferation and animal development. High eIF5A expression is observed in many tumor types and has been linked to cancer metastasis. Recent studies have shown that eIF5A facilitates the translation elongation of stretches of consecutive prolines. Activated eIF5A binds to the empty E-site of stalled ribosomes, where it is thought to interact with the peptidyl-tRNA situated at the P-site. Here, we report a genome-wide analysis of ribosome stalling in Saccharomyces cerevisiae eIF5A depleted cells using 5Pseq. We confirm that, in the absence of eIF5A, ribosomes stall at proline stretches, and extend previous studies by identifying eIF5A-dependent ribosome pauses at termination and at >200 tripeptide motifs. We show that presence of proline, glycine and charged amino acids at the peptidyl transferase center and at the beginning of the peptide exit tunnel arrest ribosomes in eIF5A-depleted cells. Lack of eIF5A also renders ribosome accumulation at the stop codons. Our data indicate specific protein functional groups under the control of eIF5A, including ER-coupled translation and GTPases in yeast and cytoskeleton organization, collagen metabolism and cell differentiation in humans. Our results support a broad mRNA-specific role of eIF5A in translation and identify the conserved motifs that affect translation elongation from yeast to humans.

Impact of high pH stress on yeast gene expression: A comprehensive analysis of mRNA turnover during stress responses

Environmental alkalinisation represents a stress condition for yeast Saccharomyces cerevisiae, to which this organism responds with extensive gene expression remodelling. We show here that alkaline pH causes an overall decrease in the transcription rate (TR) and a fast destabilisation of mRNAs, followed by a more prolonged stabilisation phase. In many cases, augmented mRNA levels occur without the TR increasing, which can be attributed to mRNA stabilisation. In contrast, the reduced amount of mRNAs is contributed by both a drop in the TR and mRNA stability. A comparative analysis with other forms of stress shows that, unlike high pH stress, heat-shock, osmotic and oxidative stresses present a common transient increase in the TR. An analysis of environmentally-responsive (ESR) genes for the four above stresses suggests that up-regulated genes are governed mostly by TR changes and complex transient bidirectional changes in mRNA stability, whereas the down-regulated ESR gene set is driven by mRNA destabilisation and a lowered TR. In all the studied forms of stress, mRNA stability plays an important role in ESR. Overall, changes in mRNA levels do not closely reflect the rapid changes in the TR and stability upon exposure to stress, which highlights the existence of compensatory mechanisms.

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.

External conditions inversely change the RNA polymerase II elongation rate and density in yeast

Elongation speed is a key parameter in RNA polymerase II (RNA pol II) activity. It affects the transcription rate, while it is conditioned by the physicochemical environment it works in at the same time. For instance, it is well-known that temperature affects the biochemical reactions rates. Therefore in free-living organisms that are able to grow at various environmental temperatures, such as the yeast Saccharomyces cerevisiae, evolution should have not only shaped the structural and functional properties of this key enzyme, but should have also provided mechanisms and pathways to adapt its activity to the optimal performance required. We studied the changes in RNA pol II elongation speed caused by alternations in growth temperature in yeast to find that they strictly follow the Arrhenius equation, and that they also provoke an almost inverse proportional change in RNA pol II density within the optimal growth temperature range (26-37 °C). Moreover, we discovered that yeast cells control the transcription initiation rate by changing the total amount of available RNA pol II.

Fertility and Polarized Cell Growth Depends on eIF5A for Translation of Polyproline-Rich Formins in Saccharomyces cerevisiae

eIF5A is an essential and evolutionary conserved translation elongation factor, which has recently been proposed to be required for the translation of proteins with consecutive prolines. The binding of eIF5A to ribosomes occurs upon its activation by hypusination, a modification that requires spermidine, an essential factor for mammalian fertility that also promotes yeast mating. We show that in response to pheromone, hypusinated eIF5A is required for shmoo formation, localization of polarisome components, induction of cell fusion proteins, and actin assembly in yeast. We also show that eIF5A is required for the translation of Bni1, a proline-rich formin involved in polarized growth during shmoo formation. Our data indicate that translation of the polyproline motifs in Bni1 is eIF5A dependent and this translation dependency is lost upon deletion of the polyprolines. Moreover, an exogenous increase in Bni1 protein levels partially restores the defect in shmoo formation seen in eIF5A mutants. Overall, our results identify eIF5A as a novel and essential regulator of yeast mating through formin translation. Since eIF5A and polyproline formins are conserved across species, our results also suggest that eIF5A-dependent translation of formins could regulate polarized growth in such processes as fertility and cancer in higher eukaryotes.

Dissection of the elements of osmotic stress response transcription factor Hot1 involved in the interaction with MAPK Hog1 and in the activation of transcription.

The response to hyperosmotic stress is mediated by the HOG pathway. The MAP kinase Hog1 activates several transcription factors, regulates chromatin-modifying enzymes and, through its interaction with RNA polymerase II, it directs this enzyme to osmotic stress-controlled genes. For such targeting, this kinase requires the interaction with transcription factors Hot1 and Sko1. However, phosphorylation of these proteins by Hog1 is not required for their functionality. In this study, we aim to identify the Hot1 elements involved in Hog1-binding and in the activation of transcription. Two-hybrid experiments demonstrated that the Hot1 sequence between amino acids 340 and 534 and the CD element of Hog1 are required for the interaction between the two proteins and the Hot1-dependent transcription regulation. Inside this Hot1 region, short sequence KRRRR (KR4, amino acids 381-385) is essential for the kinase binding. Our data show that another element, sequence EDDDDD (ED5, amino acids 541-546), is essential for Hot1 binding to chromatin. Under osmotic stress conditions, both Hot1 elements, Hog1-interaction KR4 and DNA-binding ED5, are involved in the appropriate recruitment of Hog1 and RNA polymerase II to genes controlled by this transcription factor. Moreover, both sequences are required for osmotolerance and KR4 is necessary for the functionality of the HOG pathway. According to several experiments described in this study, the Hot1 protein is capable of forming homodimers.