Schizosaccharomyces pombe RNA polymerase II comprises twelve different subunits. Its Rpb9 subunit comprises 113 amino acids, and is the only non-essential subunit of S. pombe RNA polymerase II. However, its functions have not been studied in S. pombe. The results presented in this study demonstrate that Rpb9 is involved in regulating growth under optimum and certain stress conditions in S. pombe. To further address the role (s) of various domains of this subunit in regulating these phenotypes, deletion mutant analysis was done. We observed that the region spanning 1-74 amino acids, encompassing the amino-terminal zinc finger domain and the linker region of Rpb9 was able to rescue the phenotypes associated with rpb9⁺deletion. We also demonstrate that the functions of this subunit are only partially conserved among yeast and humans. Our computational biology approaches provide a structural basis for the differential role of various Rpb9 domains in S. pombe. Furthermore, using these tools we show that there has been a co-evolution of the interaction residues between the Rpb9 subunit and the two largest subunits of RNA polymerase II, allowing for a more stringent organism-specific packing. Taken together, our results have provided functional and structural insights into the Rpb9 subunit of S. pombe.

RNA polymerase II is a conserved multi-subunit enzyme made up of twelve different subunits. Two of these subunits, Rpb4 and Rpb7, have been shown to perform functions in both transcription as well as outside of transcription in Saccharomyces cerevisiae. However, our knowledge about the roles of these subunits in Schizosaccharomyces pombe and higher eukaryotes is still limited. Moreover, both Rpb4 and Rpb7 are indispensable for viability of S. pombe and higher eukaryotes, in comparison to S. cerevisiae where deletion of only Rpb7 results in lethality. Therefore in this study, we used S. pombe strains expressing reduced levels of these subunits to determine their impact on the S. pombe proteome employing i-TRAQ based proteomics approach. Furthermore, proteomic profiling was carried out at two different time points to gain a temporal insight into the processes regulated by Rpb4 and Rpb7. The results showed that reduced levels of either Rpb4 or Rpb7 affected the expression of proteins involved in metabolism and ribosome biogenesis at both the time points. Our polysomal profiling experiments further revealed a role of these subunits in translation. Taken together, our results suggest a key role of Rpb4 and Rpb7 subunits in ribosome biogenesis and protein translation in S. pombe. SIGNIFICANCE: Rpb4 and Rpb7 subunits of RNA polymerase II are known for their diverse roles in regulating transcription, mRNA export, mRNA decay, stress response and translation in S. cerevisiae. However, their roles in other organisms are yet to be characterized in detail. Different lines of evidence also suggest that these subunits may function independently as well as a complex in budding yeast. Therefore, in the present study we employed a genome-wide quantitative proteomics-based approach to gain deeper insights into their cellular roles, and to examine if they regulate similar or different biological pathways in fission yeast. Our results provide evidence that they are both involved in primarily regulating metabolic pathways and ribosome biogenesis and also, play a role in protein translation in S. pombe.

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module.

The evolutionarily conserved RNA Polymerase II Rpb4/7 sub-complex has been thoroughly studied in yeast and impacts gene expression at multiple levels including transcription, mRNA processing and decay. In addition Rpb4/7 exerts differential effects on gene expression in yeast and Rpb4 is not obligatory for yeast (S. cerevisiae) survival. Specialised roles for human (hs) Rpb4/7 have not been extensively described and we have probed this question by depleting hsRpb4/7 in established human cell lines using RNA interference. We find that Rpb4/7 protein levels are inter-dependent and accordingly, the functional effects of depleting either protein are co-incident. hsRpb4/7 exhibits gene-specific effects and cells initially remain viable upon hsRpb4/7 depletion. However prolonged hsRpb4/7 depletion is cytotoxic in the range of cell lines tested. Protracted cell death occurs by an unknown mechanism and in some cases is accompanied by a pronounced elongated cell morphology. In conclusion we provide evidence for a gene-specific role of hsRpb4/7 in human cell viability.

Cell size is highly variable; cells from various tissues differ in volume over orders of magnitudes, from tiny lymphocytes to giant neurons, and cells of a given type change size during the cell cycle. Larger cells need to produce and maintain higher amounts of RNA and protein to sustain biomass and function, although the genome content often remains constant. Available data indicate that the transcriptional and translational outputs scale with cell size at a genome-wide level, but how such remarkably coordinated regulation is achieved remains largely mysterious. With global and systems-level approaches becoming more widespread and quantitative, it is worth revisiting this fascinating problem. Here, we outline current knowledge of the fundamental relations between genome regulation and cell size, and highlight the biological implications and potential mechanisms of the global tuning of gene expression to cellular volume.

Rpb4, a subunit of RNA Polymerase II plays an important role in various stress responses in budding yeast, Saccharomyces cerevisiae. In response to nitrogen starvation, diploid yeast undergoes a dimorphic transition to filamentous pseudohyphal growth, which is regulated through cAMP-PKA and MAP kinase pathway. In the present study, we show that disruption of Rpb4 leads to enhanced pseudohyphal growth, which is independent of nutritional status. We observed that the rpb4Delta/rpb4Delta cells exhibit pseudohyphae even in the absence of functional MAP kinase and cAMP-PKA pathways. Genome-wide expression profiling showed that in the absence of Rpb4 several genes controlling mother daughter cell separation are down regulated. Our genetic studies also provide evidence for involvement of RNA Pol II subunit Rpb4 in the expression of genes downstream of the RAM pathway. Finally, we show that this effect on expression of RAM pathway may at least be partially responsible for the pseudohyphal phenotype of rpb4Delta/rpb4Delta cells.

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.

The halophilic archaeon Haloferax volcanii encodes two related proteasome-activating nucleotidase proteins, PanA and PanB, with PanA levels predominant during all phases of growth. In this study, an isogenic panA mutant strain of H. volcanii was generated. The growth rate and cell yield of this mutant strain were lower than those of its parent and plasmid-complemented derivatives. In addition, a consistent and discernible 2.1-fold increase in the number of phosphorylated proteins was detected when the panA gene was disrupted, based on phosphospecific fluorescent staining of proteins separated by 2-dimensional gel electrophoresis. Subsequent enrichment of phosphoproteins by immobilized metal ion and metal oxide affinity chromatography (in parallel and sequentially) followed by tandem mass spectrometry was employed to identify key differences in the proteomes of these strains as well as to add to the restricted numbers of known phosphoproteins within the Archaea. In total, 625 proteins (approximately 15% of the deduced proteome) and 9 phosphosites were identified by these approaches, and 31% (195) of the proteins were identified by multiple phosphoanalytical methods. In agreement with the phosphostaining results, the number of identified proteins that were reproducibly exclusive or notably more abundant in one strain was nearly twofold greater for the panA mutant than for the parental strain. Enriched proteins exclusive to or more abundant in the panA mutant (versus the wild type) included cell division (FtsZ, Cdc48), dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase system (EI, DhaK), and oxidoreductase homologs. Differences in transcriptional regulation and signal transduction proteins were also observed, including those differences (e.g., OsmC and BolA) which suggest that proteasome deficiency caused an up-regulation of stress responses (e.g., OsmC versus BolA). Consistent with this, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems were enriched and identified as unique to the panA mutant. The cumulative proteomic data not only furthered our understanding of the archaeal proteasome system but also facilitated the assembly of the first subproteome map of H. volcanii.

Cell division is controlled by a complex network involving regulated transcription of genes and postranslational modification of proteins. The aim of this study is to demonstrate that the Mediator complex, a general regulator of transcription, is involved in the regulation of the second phase (cell separation) of cell division of the fission yeast Schizosaccharomyces pombe. In previous studies we have found that the fission yeast cell separation genes sep10 ( + ) and sep15 ( + ) code for proteins (Med31 and Med8) associated with the Mediator complex. Here, we show by genome-wide gene expression profiling of mutants defective in these genes that both Med8 and Med31 control large, partially overlapping sets of genes scattered over the entire genome and involved in diverse biological functions. Six cell separation genes controlled by the transcription factors Sep1 and Ace2 are among the target genes. Since neither sep1 ( + ) nor ace2 ( + ) is affected in the mutant cells, we propose that the Med8 and Med31 proteins act as coactivators of the Sep1-Ace2-dependent cell separation genes. The results also indicate that the subunits of Mediator may contribute to the coordination of cellular processes by fine-tuning of the expression of larger sets of genes.

In cell-walled organisms, a cross wall (septum) is produced during cytokinesis, which then splits in certain organisms to allow the daughter cells to separate. The formation and the subsequent cleavage of the septum require wall synthesis and wall degradation, which need to be strictly coordinated in order to prevent cell lysis. The dividing fission yeast (Schizosaccharomyces) cell produces a three-layered septum in which the middle layer and a narrow band of the adjacent cell wall can be degraded without threatening the integrity of the separating daughter cells. This spatially very precise process requires the activity of the Agn1p 1,3-alpha-glucanase and the Eng1p 1,3-beta-glucanase, which are localized to the septum by a complex mechanism involving the formation of a septin ring and the directed activity of the exocyst system. The Sep1p-Ace2p transcription-factor cascade regulates the expression of many genes producing proteins for this complex process. Recent advances in research into the molecular mechanisms of separation and its regulation are discussed in this review.