Rpb4 and Puf3 imprint and post-transcriptionally control the stability of a common set of mRNAs in yeast.

Gene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, crosstalk in which mRNA decay machinery and transcription machinery respectively impact transcription and mRNA stability. Rpb4, and likely dimer Rpb4/7, seem the central components of the RNA pol II governing these processes. In this work we unravel the molecular mechanisms participated by Rpb4 that mediate the posttranscriptional events regulating mRNA imprinting and stability. By RIP-Seq, we analysed genome-wide the association of Rpb4 with mRNAs and demonstrated that it targeted a large population of more than 1400 transcripts. A group of these mRNAs was also the target of the RNA binding protein, Puf3. We demonstrated that Rpb4 and Puf3 physically, genetically, and functionally interact and also affect mRNA stability, and likely the imprinting, of a common group of mRNAs. Furthermore, the Rpb4 and Puf3 association with mRNAs depends on one another. We also demonstrated, for the first time, that Puf3 associates with chromatin in an Rpb4-dependent manner. Our data also suggest that Rpb4 could be a key element of the RNA pol II that coordinates mRNA synthesis, imprinting and stability in cooperation with RBPs.

Rpb1 foot mutations demonstrate a major role of Rpb4 in mRNA stability during stress situations in yeast.

The RPB1 mutants in the foot region of RNA polymerase II affect the assembly of the complex by altering the correct association of both the Rpb6 and the Rpb4/7 dimer. Assembly defects alter both transcriptional activity as well as the amount of enzyme associated with genes. Here, we show that the global transcriptional analysis of foot mutants reveals the activation of an environmental stress response (ESR), which occurs at a permissive temperature under optimal growth conditions. Our data indicate that the ESR that occurs in foot mutants depends mostly on a global post-transcriptional regulation mechanism which, in turn, depends on Rpb4-mRNA imprinting. Under optimal growth conditions, we propose that Rpb4 serves as a key to globally modulate mRNA stability as well as to coordinate transcription and decay. Overall, our results imply that post-transcriptional regulation plays a major role in controlling the ESR at both the transcription and mRNA decay levels.

CYGD: the Comprehensive Yeast Genome Database.

The Comprehensive Yeast Genome Database (CYGD) compiles a comprehensive data resource for information on the cellular functions of the yeast Saccharomyces cerevisiae and related species, chosen as the best understood model organism for eukaryotes. The database serves as a common resource generated by a European consortium, going beyond the provision of sequence information and functional annotations on individual genes and proteins. In addition, it provides information on the physical and functional interactions among proteins as well as other genetic elements. These cellular networks include metabolic and regulatory pathways, signal transduction and transport processes as well as co-regulated gene clusters. As more yeast genomes are published, their annotation becomes greatly facilitated using S.cerevisiae as a reference. CYGD provides a way of exploring related genomes with the aid of the S.cerevisiae genome as a backbone and SIMAP, the Similarity Matrix of Proteins. The comprehensive resource is available under http://mips.gsf.de/genre/proj/yeast/.

Statistical analysis of yeast genomic downstream sequences reveals putative polyadenylation signals.

The study of a few genes has permitted the identification of three elements that constitute a yeast polyadenyl-ation signal: the efficiency element (EE), the positioning element and the actual site for cleavage and poly-adenyl-ation. In this paper we perform an analysis of oligonucleotide composition on the sequences located downstream of the stop codon of all yeast genes. Several oligonucleotide families appear over-represented with a high significance (referred to herein as 'words'). The family with the highest over-representation includes the oligonucleotides shown experimentally to play a role as EEs. The word with the highest score is TATATA, followed, among others, by a series of single-nucleotide variants (TATGTA, TACATA, TAAATA.) and one-letter shifts (ATATAT). A position analysis reveals that those words have a high preference to be in 3' flanks of yeast genes and there they have a very uneven distribution, with a marked peak around 35 bp after the stop codon. Of the predicted ORFs, 85% show one or more of those sequences. Similar results were obtained using a data set of EST sequences. Other clusters of over-represented words are also detected, namely T- and A-rich signals. Using these results and previously known data we propose a general model for the 3' trailers of yeast mRNAs.

Sliding-end-labelling. A method to avoid artifacts in nucleosome positioning.

A method, termed 'sliding-end-labelling', has been devised to avoid a frequent artifact in nucleosome positioning by indirect end labelling, namely the appearing of DNA fragments originated by two nuclease cuts, one of them lying within the region covered by the probe. The method is applied to the nucleosome positioning in the yeast SUC2 gene for invertase.

On the presence of HMG proteins in yeast.

Two polypeptides antigenically related to mammalian HMG1/2 have been detected in the yeast Saccharomyces cerevisiae. One exhibits an electrophoretic mobility in the range of mammalian HMG1/2 whereas the second polypeptide comigrates with yeast HMG S4. Evidence for the presence of HMG14/17 in yeast has not been obtained by immunological or nuclease digestion-based methods, although their presence cannot be excluded.

Bromodomain factor 1 (Bdf1) protein interacts with histones.

Using a yeast two-hybrid assay we detected an interaction between the N-terminal region of histone H4 (amino acids 1--59) and a fragment of the bromodomain factor 1 protein (Bdf1p) (amino acids 304--571) that includes one of the two bromodomains of this protein. No interaction was observed using fragments of histone H4 sequence smaller than the first 59 amino acids. Recombinant Bdf1p (rBdf1p) demonstrates binding affinity for histones H4 and H3 but not H2A and H2B in vitro. Moreover, rBdf1p is able to bind histones H3 and H4 having different degrees of acetylation. Finally, we have not detected histone acetyltransferase activity associated with Bdf1p.

Expression levels and patterns of glycolytic yeast genes during wine fermentation.

Promoters from glycolytic genes are widely used for gene overexpression in the yeast Saccharomyces cerevisiae. Wine strains are not an exception, and genes of enological interest have been expressed in this way. However, the transcriptional pattern of glycolytic genes has never been studied during wine fermentation. In this work we describe the levels and expression patterns of glycolytic genes for a wine yeast strain during the alcoholic fermentation of three different musts. Results show similar transcriptional patterns for all genes studied: maximal levels of mRNA at the exponential growth stage, and a gradual decrease during the stationary phase, in accordance with the fermentation rates. The glyceraldehyde 3-phosphate dehydrogenase genes reach the highest transcriptional levels during wine fermentation, similarly as previously described for laboratory strains and conditions.

Transcriptional response of Saccharomyces cerevisiae to different nitrogen concentrations during alcoholic fermentation.

Gene expression profiles of a wine strain of Saccharomyces cerevisiae PYCC4072 were monitored during alcoholic fermentations with three different nitrogen supplies: (i) control fermentation (with enough nitrogen to complete sugar fermentation), (ii) nitrogen-limiting fermentation, and (iii) the addition of nitrogen to the nitrogen-limiting fermentation (refed fermentation). Approximately 70% of the yeast transcriptome was altered in at least one of the fermentation stages studied, revealing the continuous adjustment of yeast cells to stressful conditions. Nitrogen concentration had a decisive effect on gene expression during fermentation. The largest changes in transcription profiles were observed when the early time points of the N-limiting and control fermentations were compared. Despite the high levels of glucose present in the media, the early responses of yeast cells to low nitrogen were characterized by the induction of genes involved in oxidative glucose metabolism, including a significant number of mitochondrial associated genes resembling the yeast cell response to glucose starvation. As the N-limiting fermentation progressed, a general downregulation of genes associated with catabolism was observed. Surprisingly, genes encoding ribosomal proteins and involved in ribosome biogenesis showed a slight increase during N starvation; besides, genes that comprise the RiBi regulon behaved distinctively under the different experimental conditions. Here, for the first time, the global response of nitrogen-depleted cells to nitrogen addition under enological conditions is described. An important gene expression reprogramming occurred after nitrogen addition; this reprogramming affected genes involved in glycolysis, thiamine metabolism, and energy pathways, which enabled the yeast strain to overcome the previous nitrogen starvation stress and restart alcoholic fermentation.

Saccharomyces cerevisiae signature genes for predicting nitrogen deficiency during alcoholic fermentation.

Genome-wide analysis of the wine yeast strain Saccharomyces cerevisiae PYCC4072 identified 36 genes highly expressed under conditions of low or absent nitrogen in comparison with a nitrogen-replete condition. Reverse transcription-PCR analysis for four of these transcripts with this strain and its validation with another wine yeast strain underlines the usefulness of these signature genes for predicting nitrogen deficiency and therefore the diagnosis of wine stuck/sluggish fermentations.

Stress response and expression patterns in wine fermentations of yeast genes induced at the diauxic shift.

During wine fermentation yeasts quickly reach a stationary phase, where cells are metabolically active by consuming sugars present in grape must. It is, consequently, of great interest at this stage to identify suitable gene promoters that may be used to induce the expression of genes with enological applications. With this aim, we have studied a group of genes showing an induction peak at the diauxic shift, and possessing stress response elements (STRE) at their promoters. We have determined their induction levels under individualized stress conditions, such as carbon source starvation or high salt concentrations. In all the cases studied, the activation and/or basal transcription are dependent on the transcriptional factors Msn2p and Msn4p. We have analysed the expression patterns and mRNA levels during wine fermentation, and have found that they are all activated at the stationary phase. Finally, we have identified SPI1, a new highly expressed yeast gene which is specifically induced at the stationary phase of both microvinification and laboratory growth conditions.

A natural A/T-rich sequence from the yeast FBP1 gene exists as a cruciform in Escherichia coli cells.

Palindromic or semipalindromic sequences can adopt cruciform structures in DNA in vitro. It has been demonstrated in some cases that A/T-rich cruciforms exist also in vivo in Escherichia coli. The biological function of those structures is not understood although putative cruciforms have been found in interesting locations on replication origins, operators, or transcriptional termination regions. Here we show by means of the use of structure-dependent nucleases that the 3' end of the yeast FBP1 gene contains a stable cruciform both in vitro and in E. coli cells and that in both cases, its extrusion depends on the DNA supercoiling state.

Chromatin structure of the yeast SUC2 promoter in regulatory mutants.

We have previously suggested that two positioned nucleosomes are removed from the promoter of the Saccharomyces cerevisiae SUC2 gene upon depression by glucose starvation. To gain further insight into the changes accompanying derepression at the chromatin level we have studied the chromatin structure of the SUC2 promoter in several mutants affecting SUC2 expression. The non-derepressible mutants snf1, snf2 and snf5 present a chromatin structure characteristic of the repressed state, irrespective of the presence or absence of glucose. The non-repressible mutants, mig1 and ssn6, as well as the double mutant snfs sn6 exhibit an opened chromatin structure even in the presence of glucose. These results suggest that the DNA-binding protein encoded by MIG1 is necessary to produce the characteristic pattern of repressed chromatin and that the SNF1 protein kinase is sufficient to produce the derepressed chromatin pattern. A model is presented for the transitions that result in opening up of the chromatin structure.

Transcriptional and structural study of a region of two convergent overlapping yeast genes.

The exceptionally close packing of many yeast genes and other chromosomal elements raises the question of how those elements are functionally insulated. All published work shows that natural insulators are very effective, but transcriptional interference (TI) occurs if they are mutated or if their natural context is altered. Mechanisms to avoid TI are poorly understood, but are thought to involve an interplay of cis sequences and trans factors in a chromatin context. We have studied the case of two convergent closely packed ORFs (56 bp of separation) in chromosome IX of Saccharomyces cerevisiae. mRNAs from POT1 and YIL161w overlap by up to 115 nt. Convergent transcription causes a small but noticeable negative effect on the level of POT1 mRNA and nucleosome displacement in the intergenic region. This suggests for the first time that some TI could occur in convergently transcribed yeast genes, even in a natural chromosomal context.

Stochastic nucleosome positioning in a yeast chromatin region is not dependent on histone H1.

To gain a better understanding of the function of the yeast histone H1, its role in nucleosome positioning was studied. With this objective in mind, we analyzed a chromatin region of the yeast Chromosome (Chr) IX, in which there are two closely packed open reading frames (ORFs), POT1 and YIL161w. This locus shows a regular ladder of 13 stochastically positioned nucleosomes, which is unaffected by the absence of the HHO1 gene. This suggests that histone H1 has no effect on nucleosome positioning in yeast.

Functional analysis of 12 ORFs from Saccharomyces cerevisiae chromosome II.

Twelve different ORFs have been deleted from the right arm of Saccharomyces cerevisiae chromosome II; namely YBR193c, YBR194w, YBR197c, YBR198c, YBR201w, YBR203w, YBR207w, YBR209w, YBR210w, YBR211c, YBR217w and YBR228w. Tetrad analysis of heterozygous deletant strains revealed that YBR193c, YBR198c and YBR211c are essential genes for vegetative growth. No effects were detected in any of the haploid deletion mutants for the rest of the ORFs with respect to growth, gross morphology or mating.

The role of histones and their modifications in the informative content of chromatin.

It is traditionally accepted that the DNA sequence cannot by itself explain all the mechanisms necessary for the development of living beings, especially in eukaryotes. Indeed part of the information used in these processes is stored in other ways, generally called 'epigenetic', whose molecular mechanisms are mostly unknown. The ultimate explanation for them might reside in the non-DNA moiety of chromatin which may play an active role in heredity ('chromatin information'). Histones are the universal structural component of chromatin. However, recent studies strongly suggest that histones, and their modifications--especially the reversible acetylation of lysines--may act as a recognition signal for regulatory proteins and they may participate, for this reason, in gene regulation. This type of information could be maintained through its replication and, ultimately, it could form the molecular basis of certain processes related to the development of the eukaryotic organisms.

An inverse correlation between stress resistance and stuck fermentations in wine yeasts. A molecular study.

During alcoholic fermentations yeast cells are subjected to several stress conditions and, therefore, yeasts have developed molecular mechanisms in order to resist this adverse situation. The mechanisms involved in stress response have been studied in Saccharomyces cerevisiae laboratory strains. However a better understanding of these mechanisms in wine yeasts could open the possibility to improve the fermentation process. In this work an analysis of the stress response in three wine yeasts has been carried out by studying the expression of several representative genes under several stress conditions which occur during fermentation. We propose a simplified method to study how these stress conditions affect the viability of yeast cells. Using this approach an inverse correlation between stress-resistance and stuck fermentations has been found. We also have preliminary data about the use of the HSP12 gene as a molecular marker for stress-resistance in wine yeasts.

Partial purification and properties of two histone acetyltransferases from the yeast, Saccharomyces cerevisiae.

Two histone acetyltransferases, A and B, have been extracted and partially purified from yeast cells. The purification scheme included ammonium sulfate precipitation, and chromatography on DEAE-Sepharose and Sephadex G-200. The basic properties of both enzymes closely correspond to those of acetyltransferase A and B found in higher eucaryotes. Yeast enzyme A elutes from DEAE-Sepharose prior to acetyltransferase B, and it is activated by low concentrations of DNA and strongly inhibited by p-chloromercuribenzoate (PCMB). Enzyme B is inhibited by DNA over the entire range of concentrations tested and it is less sensitive to PCMB than enzyme A. When assayed with yeast whole histones, enzyme B shows a marked specificity toward histone H4, although H3 and H2B are also accepted as substrates. Enzyme A preferentially catalyzes the acetylation of yeast H2B and H3, with the other two core histones being acetylated to a much lesser extent.

DNase I sensitivity of the chromatin of the yeast SUC2 gene for invertase.

The DNase I sensitivity of chromatin of the yeast SUC2 gene, which encodes two forms of invertase, has been studied both in the genome and in a multicopy plasmid carrying the gene and its flaking sequences. Whereas little if any difference in the DNase I sensitivity of the flanking regions was found between the repressed and the derepressed states, derepression of the gene was accompanied by a large increase in the sensitivity of the transcribed region. A well-defined DNase I hypersensitive site was found centered at approximately 120 bp downstream from the end of the coding region. This site seems to be flanked in the 3' non-coding region by strictly positioned nucleosomes, and the structure of this region changes upon derepression. In the 5' non-coding region two DNase I hypersensitive sites have been found flanking the TATA box and a set of three closely spaced hypersensitive sites occurs in an upstream regulatory sequence. The structure of these latter sites depends on the on-off state of transcription.