Tho2 is critical for the recruitment of Rrp6 to chromatin in response to perturbed mRNP biogenesis.

The eukaryotic THO complex coordinates the assembly of so-called messenger RNA-ribonucleoprotein particles (mRNPs), a process that involves cotranscriptional coating of nascent mRNAs with proteins. Once formed, mRNPs undergo a quality control step that marks them either for active transport to the cytoplasm, or Rrp6/RNA exosome-mediated degradation in the nucleus. However, the mechanism behind the quality control of nascent mRNPs is still unclear. We investigated the cotranscriptional quality control of mRNPs in budding yeast by expressing the bacterial Rho helicase, which globally perturbs yeast mRNP formation. We examined the genome-wide binding profiles of the THO complex subunits Tho2, Thp2, Hpr1, and Mft1 upon perturbation of the mRNP biogenesis, and found that Tho2 plays two roles. In addition to its function as a subunit of the THO complex, upon perturbation of mRNP biogenesis Tho2 targets Rrp6 to chromatin via its carboxy-terminal domain. Interestingly, other THO subunits are not enriched on chromatin upon perturbation of mRNP biogenesis and are not necessary for localizing Rrp6 at its target loci. Our study highlights the potential role of Tho2 in cotranscriptional mRNP quality control, which is independent of other THO subunits. Considering that both the THO complex and the RNA exosome are evolutionarily highly conserved, our findings are likely relevant for mRNP surveillance in mammals.

Antisense non-coding transcription represses the PHO5 model gene at the level of promoter chromatin structure.

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.

Interplay of the RNA Exosome Complex and RNA-Binding Protein Ssd1 in Maintaining Cell Wall Stability in Yeast.

Yeast cell wall stability is important for cell division and survival under stress conditions. The expression of cell-wall-related proteins is regulated by several pathways involving RNA-binding proteins and RNases. The multiprotein RNA exosome complex provides the 3'→5' exoribonuclease activity that is critical for maintaining the stability and integrity of the yeast cell wall under stress conditions such as high temperatures. In this work, we show that the temperature sensitivity of RNA exosome mutants is most pronounced in the W303 genetic background due to the nonfunctional ssd1-d allele. This gene encodes the RNA-binding protein Ssd1, which is involved in the posttranscriptional regulation of cell-wall-related genes. Expression of the functional SSD1-V allele from its native genomic locus or from a centromeric plasmid suppresses the growth defects and aberrant morphology of RNA exosome mutant cells at high temperatures or upon treatment with cell wall stressors. Moreover, combined inactivation of the RNA exosome catalytic subunit Rrp6 and Ssd1 results in a synthetically sick phenotype of cell wall instability, as these proteins may function in parallel pathways (i.e., via different mRNA targets) to maintain cell wall stability. IMPORTANCE Stressful conditions such as high temperatures can compromise cellular integrity and cause bursting. In microorganisms surrounded by a cell wall, such as yeast, the cell wall is the primary shield that protects cells from environmental stress. Therefore, remodeling its structure requires inputs from multiple signaling pathways and regulators. In this work, we identify the interplay of the RNA exosome complex and the RNA-binding protein Ssd1 as an important factor in the yeast cell wall stress response. These proteins operate in independent pathways to support yeast cell wall stability. This work highlights the contribution of RNA-binding proteins in the regulation of yeast cell wall structure, providing new insights into yeast physiology.

Regulation of the conserved 3'-5' exoribonuclease EXOSC10/Rrp6 during cell division, development and cancer.

The conserved 3'-5' exoribonuclease EXOSC10/Rrp6 processes and degrades RNA, regulates gene expression and participates in DNA double-strand break repair and control of telomere maintenance via degradation of the telomerase RNA component. EXOSC10/Rrp6 is part of the multimeric nuclear RNA exosome and interacts with numerous proteins. Previous clinical, genetic, biochemical and genomic studies revealed the protein's essential functions in cell division and differentiation, its RNA substrates and its relevance to autoimmune disorders and oncology. However, little is known about the regulatory mechanisms that control the transcription, translation and stability of EXOSC10/Rrp6 during cell growth, development and disease and how these mechanisms evolved from yeast to human. Herein, we provide an overview of the RNA- and protein expression profiles of EXOSC10/Rrp6 during cell division, development and nutritional stress, and we summarize interaction networks and post-translational modifications across species. Additionally, we discuss how known and predicted protein interactions and post-translational modifications influence the stability of EXOSC10/Rrp6. Finally, we explore the idea that different EXOSC10/Rrp6 alleles, which potentially alter cellular protein levels or affect protein function, might influence human development and disease progression. In this review we interpret information from the literature together with genomic data from knowledgebases to inspire future work on the regulation of this essential protein's stability in normal and malignant cells.

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation.

Nuclear RNA exosome is the main 3'→5' RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes.

Non-coding RNAs as cell wall regulators in Saccharomyces cerevisiae.

The cell wall of Saccharomyces cerevisiae is an extracellular organelle crucial for preserving its cellular integrity and detecting environmental cues. The cell wall is composed of mannoproteins attached to a polysaccharide network and is continuously remodelled as cells undergo cell division, mating, gametogenesis or adapt to stressors. This makes yeast an excellent model to study the regulation of genes important for cell wall formation and maintenance. Given that certain yeast strains are pathogenic, a better understanding of their life cycle is of clinical relevance. This is why transcriptional regulatory mechanisms governing genes involved in cell wall biogenesis or maintenance have been the focus of numerous studies. However, little is known about the roles of long non-coding RNAs (lncRNAs), a class of transcripts that are thought to possess little or no protein coding potential, in controlling the expression of cell wall-related genes. This review outlines currently known mechanisms of lncRNA-mediated regulation of gene expression in S. cerevisiae and describes examples of lncRNA-regulated genes encoding cell wall proteins. We suggest that the association of currently annotated lncRNAs with the coding sequences and/or promoters of cell wall-related genes highlights a potential role for lncRNAs as important regulators of the yeast cell wall structure.

The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast.

5-fluorouracil (5-FU) was isolated as an inhibitor of thymidylate synthase, which is important for DNA synthesis. The drug was later found to also affect the conserved 3'-5' exoribonuclease EXOSC10/Rrp6, a catalytic subunit of the RNA exosome that degrades and processes protein-coding and non-coding transcripts. Work on 5-FU's cytotoxicity has been focused on mRNAs and non-coding transcripts such as rRNAs, tRNAs and snoRNAs. However, the effect of 5-FU on long non-coding RNAs (lncRNAs), which include regulatory transcripts important for cell growth and differentiation, is poorly understood. RNA profiling of synchronized 5-FU treated yeast cells and protein assays reveal that the drug specifically inhibits a set of cell cycle regulated genes involved in mitotic division, by decreasing levels of the paralogous Swi5 and Ace2 transcriptional activators. We also observe widespread accumulation of different lncRNA types in treated cells, which are typically present at high levels in a strain lacking EXOSC10/Rrp6. 5-FU responsive lncRNAs include potential regulatory antisense transcripts that form double-stranded RNAs (dsRNAs) with overlapping sense mRNAs. Some of these transcripts encode proteins important for cell growth and division, such as the transcription factor Ace2, and the RNA exosome subunit EXOSC6/Mtr3. In addition to revealing a transcriptional effect of 5-FU action via DNA binding regulators involved in cell cycle progression, our results have implications for the function of putative regulatory lncRNAs in 5-FU mediated cytotoxicity. The data raise the intriguing possibility that the drug deregulates lncRNAs/dsRNAs involved in controlling eukaryotic cell division, thereby highlighting a new class of promising therapeutical targets.

In the quest for new targets for pathogen eradication: the adenylosuccinate synthetase from the bacterium Helicobacter pylori.

Adenylosuccinate synthetase (AdSS) is an enzyme at regulatory point of purine metabolism. In pathogenic organisms which utilise only the purine salvage pathway, AdSS asserts itself as a promising drug target. One of these organisms is Helicobacter pylori, a wide-spread human pathogen involved in the development of many diseases. The rate of H. pylori antibiotic resistance is on the increase, making the quest for new drugs against this pathogen more important than ever. In this context, we describe here the properties of H. pylori AdSS. This enzyme exists in a dimeric active form independently of the presence of its ligands. Its narrow stability range and pH-neutral optimal working conditions reflect the bacterium's high level of adaptation to its living environment. Efficient inhibition of H. pylori AdSS with hadacidin and adenylosuccinate gives hope of finding novel drugs that aim at eradicating this dangerous pathogen.

Proteolytic processing of the Saccharomyces cerevisiae cell wall protein Scw4 regulates its activity and influences its covalent binding to glucan.

Yeast cell wall contains a number of proteins that are either non-covalently (Scw-proteins), or covalently (Ccw-proteins) bound to ß-1,3-glucan, the latter either through GPI-anchors and ß-1,6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). It was shown that a part of Scw4, previously identified among the non-covalently bound cell wall proteins, was covalently attached to wall polysaccharides by a so far unknown alkali sensitive linkage. Thus Scw4 could be released from cell walls by treatments with hot SDS, mild alkali, or ß-1,3-glucanases, respectively. It was further shown that non-covalently bound Scw4 (SDS released) underwent the Kex2 proteolytic processing. In this paper it was demonstrated that Scw4 was also processed by yapsins at a position 9 amino acids downstream of the Kex2 cleavage site. Scw4 cleaved at the yapsin site had a markedly lower potential for covalent attachment to glucan. The overproduction of the fully processed form of Scw4 lead to high mortality, particularly in the stationary phase of growth, and to markedly increased cell size. On the other hand, the overproduction of Scw4 processed only by Kex2 or not processed at all had no apparent change in mortality indicating that only the smallest, completely mature form of Scw4 had the activity leading to observed phenotype changes.

The epigenetic processes of meiosis in male mice are broadly affected by the widely used herbicide atrazine.

BACKGROUND: Environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice. METHODS: Gene expression pattern was analysed by Gene-Chip array. Genome-wide mapping of H3K4me3 marks distribution was done by ChIP-sequencing of testis tissue using Illumina technologies. RT-qPCR was used to validate differentially expressed genes or differential peaks. RESULTS: We demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome. CONCLUSIONS: Our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.

The histone deacetylase Rpd3/Sin3/Ume6 complex represses an acetate-inducible isoform of VTH2 in fermenting budding yeast cells.

The tripartite Rpd3/Sin3/Ume6 complex represses meiotic isoforms during mitosis. We asked if it also controls starvation-induced isoforms. We report that VTH1/VTH2 encode acetate-inducible isoforms with extended 5'-regions overlapping antisense long non-coding RNAs. Rpd3 and Ume6 repress the long isoform of VTH2 during fermentation. Cells metabolising glucose contain Vth2, while the protein is undetectable in acetate and during sporulation. VTH2 is a useful model locus to study mechanisms implicating promoter directionality, lncRNA transcription and post-transcriptional control of gene expression via 5'-UTRs. Since mammalian genes encode transcript isoforms and Rpd3 is conserved, our findings are relevant for gene expression in higher eukaryotes.

Integrated RNA- and protein profiling of fermentation and respiration in diploid budding yeast provides insight into nutrient control of cell growth and development.

Diploid budding yeast undergoes rapid mitosis when it ferments glucose, and in the presence of a non-fermentable carbon source and the absence of a nitrogen source it triggers sporulation. Rich medium with acetate is a commonly used pre-sporulation medium, but our understanding of the molecular events underlying the acetate-driven transition from mitosis to meiosis is still incomplete. We identified 263 proteins for which mRNA and protein synthesis are linked or uncoupled in fermenting and respiring cells. Using motif predictions, interaction data and RNA profiling we find among them 28 likely targets for Ume6, a subunit of the conserved Rpd3/Sin3 histone deacetylase-complex regulating genes involved in metabolism, stress response and meiosis. Finally, we identify 14 genes for which both RNA and proteins are detected exclusively in respiring cells but not in fermenting cells in our sample set, including CSM4, SPR1, SPS4 and RIM4, which were thought to be meiosis-specific. Our work reveals intertwined transcriptional and post-transcriptional control mechanisms acting when a MATa/α strain responds to nutritional signals, and provides molecular clues how the carbon source primes yeast cells for entering meiosis. BIOLOGICAL SIGNIFICANCE: Our integrated genomics study provides insight into the interplay between the transcriptome and the proteome in diploid yeast cells undergoing vegetative growth in the presence of glucose (fermentation) or acetate (respiration). Furthermore, it reveals novel target genes involved in these processes for Ume6, the DNA binding subunit of the conserved histone deacetylase Rpd3 and the co-repressor Sin3. We have combined data from an RNA profiling experiment using tiling arrays that cover the entire yeast genome, and a large-scale protein detection analysis based on mass spectrometry in diploid MATa/α cells. This distinguishes our study from most others in the field-which investigate haploid yeast strains-because only diploid cells can undergo meiotic development in the simultaneous absence of a non-fermentable carbon source and nitrogen. Indeed, we report molecular clues how respiration of acetate might prime diploid cells for efficient spore formation, a phenomenon that is well known but poorly understood.

The conserved histone deacetylase Rpd3 and the DNA binding regulator Ume6 repress BOI1's meiotic transcript isoform during vegetative growth in Saccharomyces cerevisiae.

BOI1 and BOI2 are paralogs important for the actin cytoskeleton and polar growth. BOI1 encodes a meiotic transcript isoform with an extended 5'-untranslated region predicted to impair protein translation. It is, however, unknown how the isoform is repressed during mitosis, and if Boi1 is present during sporulation. By interpreting microarray data from MATa cells, MATa/α cells, a starving MATα/α control, and a meiosis-impaired rrp6 mutant, we classified BOI1's extended isoform as early meiosis-specific. These results were confirmed by RNA-Sequencing, and extended by a 5'-RACE assay and Northern blotting, showing that meiotic cells induce the long isoform while the mitotic isoform remains detectable during meiosis. We provide evidence via motif predictions, an in vivo binding assay and genetic experiments that the Rpd3/Sin3/Ume6 histone deacetylase complex, which represses meiotic genes during mitosis, also prevents the induction of BOI1's 5'-extended isoform in mitosis by direct binding of Ume6 to its URS1 target. Finally, we find that Boi1 protein levels decline when cells switch from fermentation to respiration and sporulation. The histone deacetylase Rpd3 is conserved, and eukaryotic genes frequently encode transcripts with variable 5'-UTRs. Our findings are therefore relevant for regulatory mechanisms involved in the control of transcript isoforms in multi-cellular organisms.

The conserved histone deacetylase Rpd3 and its DNA binding subunit Ume6 control dynamic transcript architecture during mitotic growth and meiotic development.

It was recently reported that the sizes of many mRNAs change when budding yeast cells exit mitosis and enter the meiotic differentiation pathway. These differences were attributed to length variations of their untranslated regions. The function of UTRs in protein translation is well established. However, the mechanism controlling the expression of distinct transcript isoforms during mitotic growth and meiotic development is unknown. In this study, we order developmentally regulated transcript isoforms according to their expression at specific stages during meiosis and gametogenesis, as compared to vegetative growth and starvation. We employ regulatory motif prediction, in vivo protein-DNA binding assays, genetic analyses and monitoring of epigenetic amino acid modification patterns to identify a novel role for Rpd3 and Ume6, two components of a histone deacetylase complex already known to repress early meiosis-specific genes in dividing cells, in mitotic repression of meiosis-specific transcript isoforms. Our findings classify developmental stage-specific early, middle and late meiotic transcript isoforms, and they point to a novel HDAC-dependent control mechanism for flexible transcript architecture during cell growth and differentiation. Since Rpd3 is highly conserved and ubiquitously expressed in many tissues, our results are likely relevant for development and disease in higher eukaryotes.

Cotranscriptional recruitment of RNA exosome cofactors Rrp47p and Mpp6p and two distinct Trf-Air-Mtr4 polyadenylation (TRAMP) complexes assists the exonuclease Rrp6p in the targeting and degradation of an aberrant messenger ribonucleoprotein particle (mRNP) in yeast.

The cotranscriptional mRNA processing and packaging reactions that lead to the formation of export-competent messenger ribonucleoprotein particles (mRNPs) are under the surveillance of quality control steps. Aberrant mRNPs resulting from faulty events are retained in the nucleus with ensuing elimination of their mRNA component. The molecular mechanisms by which the surveillance system recognizes defective mRNPs and stimulates their destruction by the RNA degradation machinery are still not completely elucidated. Using an experimental approach in which mRNP formation in yeast is disturbed by the action of the bacterial Rho helicase, we have shown previously that the targeting of Rho-induced aberrant mRNPs is mediated by Rrp6p, which is recruited cotranscriptionally in association with Nrd1p following Rho action. Here we investigated the specific involvement in this quality control process of different cofactors associated with the nuclear RNA degradation machinery. We show that, in addition to the main hydrolytic action of the exonuclease Rrp6p, the cofactors Rrp47p, Mpp6p as well as the Trf-Air-Mtr4 polyadenylation (TRAMP) components Trf4p, Trf5p, and Air2p contribute significantly by stimulating the degradation process upon their cotranscriptional recruitment. Trf4p and Trf5p are apparently recruited in two distinct TRAMP complexes that both contain Air2p as component. Surprisingly, Rrp47p appears to play an important role in mutual protein stabilization with Rrp6p, which highlights a close association between the two partners. Together, our results provide an integrated view of how different cofactors of the RNA degradation machinery cooperate to target and eliminate aberrant mRNPs.

Characterization of Ccw7p cell wall proteins and the encoding genes of Saccharomyces cerevisiae wine yeast strains: relevance for flor formation.

The specific flavour of Sherry-type wines requires aromatic compounds produced as by-products of the oxidative metabolism of yeasts that are able to form a biofilm (flor) at the wine surface. A similar yeast pellicle develops on the surface of 'Tokaji Szamorodni', one of the traditional Hungarian botrytized wines, during maturation. In this work, patterns of biotinylated cell wall proteins extracted from film-forming and nonfilm-forming Saccharomyces cerevisiae strains were compared. It was found that all the tested 23 film-forming 'Szamorodni' yeast strains had a decreased size of the Ccw7/Hsp150 protein, one of the members of the Pir-protein family. Sequencing of the encoding genes revealed that the strains were lacking three out of the 11 repeating sequences characteristic to this protein family. One of the film-forming strains contained CCW7 alleles of different length, which was generated by intragenic tandem duplication of a sequence containing two repetitive domains. Unlike the film-forming strains, 16 nonfilm-forming wine yeasts isolated from a different botrytized wine, 'Tokaji Aszu', showed pronounced polymorphism of the CCW7 locus. It is highly probable that the modified Ccw7 protein does not contribute to the increased hydrophobicity of film-forming strains but it may influence molecular reorganization of the cell wall during stress adaptation.

Binding assay for incorporation of alkali-extractable proteins in the Saccharomyces cerevisiae cell wall.

Yeasts have developed three different ways of attaching proteins to cell wall glucan. Some proteins are bound to beta-1,3-glucan non-covalently, while others are attached covalently, through GPI-anchor and beta-1,6-glucan, or directly to beta-1,3-glucan by alkali-labile ester linkage between the gamma-carboxyl groups of glutamic acid and the hydroxyl groups of glucoses (Pir proteins). In order to obtain further insight into the binding mechanism, a novel, simple binding assay for Pir-family proteins was developed. It has been shown that PIR, as well as SCW4 mutants, can bind externally added Ccw5p to their cell walls. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild-type and mutant cells showed enhanced binding of the Ccw5p in 0.6 M KCl. After disruption of all Pir genes (CCW5, CCW6, CCW7 and CCW8), 67 kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant resulted in disappearance of the 67 kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall by a so-far unidentified alkali-labile linkage.

Increased mortality of Saccharomyces cerevisiae cell wall protein mutants.

The yeast cell wall contains an unusually high number of different mannoproteins. The physiological role of most of them is unknown and gene disruptions leading to depletion of different proteins do not affect major functions of the wall. In this work the phenotype of different single and multiple cell wall protein mutants was observed at the level of individual cells. It was found that the lack of the non-covalently bound wall proteins Scw4p, Scw10p and Bgl2p increases the mortality of Saccharomyces cerevisiae cells grown exponentially under standard laboratory conditions, as assayed by methylene blue staining. Mutation of SCW11, however, suppressed the phenotype of scw4scw10, or scw4scw10bgl2, indicating that Scw4p, Scw10p and Bgl2p act synergistically while Scw11p has an activity antagonistic to that of the other three proteins. Mutants lacking major covalently bound proteins, either all four described Pir-proteins or the five most abundant glycosylphosphatidylinositol (GPI)-anchored proteins (Ccw12p, Ccw13p/Dan1p, Ccw14p/Icwp1p, Tip1p and Cwp1p), also had increased mortalities, the first somewhat more and the latter less than that of scw4scw10bgl2. In all cases the observed phenotype was suppressed by the addition of an osmotic stabilizer to the growth medium, indicating that cells died due to decreased osmotic stability. If cells were grown to stationary phase, Scw-mutants showed only slightly increased mortality, but mutants lacking Pir- or GPI-anchored proteins had significantly increased sensitivity, suggesting that their physiological function is primarily expressed in stationary-phase cells. In many cases structures consisting of a living ccw5ccw6ccw7ccw8 (multiple Pir-protein mutant) mother with two methylene blue-stained daughters could be seen.