Parag P. Sadhale

Whole genome expression profiles of yeast RNA polymerase II core subunit, Rpb4, in stress and nonstress conditions

Organisms respond to environmental stress by adopting changes in gene expression at the transcriptional level. Rpb4, a nonessential subunit of the core RNA polymerase II has been proposed to play a role in non-stress-specific transcription and in the regulation of stress response in yeast. We find that in addition to the temperature sensitivity of the null mutant of Rpb4, diploid null mutants are also compromised in sporulation and show morphological changes associated with nitrogen starvation. Using whole genome expression analysis, we report here the effects of Rpb4 on expression of genes during normal growth and following heat shock and nutritional starvation. Our analysis shows that Rpb4 affects expression of a small yet significant fraction of the genome in both stress and normal conditions. We found that genes involved in galactose metabolism were dependent on the presence of Rpb4 irrespective of the environmental condition. Rpb4 was also found to affect the expression of several other genes specifically in conditions of nutritional starvation. The general defect in the absence of Rpb4 is in the expression of metabolic genes, especially those involved in carbon metabolism and energy generation. We report that various stresses are affected byRPB4 and that on overexpression the stress-specific activators can partially rescue the corresponding defects.

Multiple Mechanisms of Suppression Circumvent Transcription Defects in an RNA Polymerase Mutant

ABSTRACT Using a high-copy-number suppressor screen to obtain clues about the role of the yeast RNA polymerase II subunit RPB4 in transcription, we identified three suppressors of the temperature sensitivity resulting from deletion of the RPB4 gene (ΔRPB4). One suppressor is Sro9p, a protein related to La protein, another is the nucleosporin Nsp1p, and the third is the RNA polymerase II subunit RPB7. Suppression by RPB7 was anticipated since its interaction with RPB4 is well established both in vitro and in vivo. We examined the effect of overexpression of each suppressor gene on transcription. Interestingly, suppression of the temperature-sensitive phenotype correlates with the correction of a characteristic transcription defect of this mutant: each suppressor restored the level of promoter-specific, basal transcription to wild-type levels. Examination of the effects of the suppressors on other in vivo transcription aberrations in ΔRPB4 cells revealed significant amelioration of defects in certain inducible genes in Sro9p and RPB7, but not in Nsp1p, suppressor cells. Analysis of mRNA levels demonstrated that overexpression of each of the three suppressors minimally doubled the mRNA levels during stationary phase. However, the elevated mRNA levels in Sro9p suppressor cells appear to result from a combination of enhanced transcription and message stability. Taken together, these results demonstrate that these three proteins influence transcription and implicate Sro9p in both transcription and posttranscription events.

Rpb4 and Rpb7: A Sub‐complex Integral to Multi‐subunit RNA Polymerases Performs a Multitude of Functions

Rpb4 and Rpb7, are conserved subunits of RNA polymerase II that play important roles in stress responses such as growth at extreme temperatures, recovery from stationary phase, sporulation and pseudohyphal growth. Recent reports have shown that apart from stress response, these proteins also affect a multitude of processes including activated transcription, mRNA export, transcription coupled repair etc. We propose a model that integrates the multifarious roles of this sub‐complex. We suggest that these proteins function by modulating interactions of one or more ancillary factors with the polymerase leading to specific transcription of subsets of these genes. Preliminary experimental evidence in support of such a model is discussed. IUBMB Life, 57: 93‐102, 2005

Genomewide recruitment analysis of Rpb4, a subunit of polymerase II in Saccharomyces cerevisiae, reveals its involvement in transcription elongation.

ABSTRACT The Rpb4/Rpb7 subcomplex of yeast RNA polymerase II (Pol II) has counterparts in all multisubunit RNA polymerases from archaebacteria to higher eukaryotes. The Rpb4/7 subcomplex in Saccharomyces cerevisiae is unique in that it easily dissociates from the core, unlike the case in other organisms. The relative levels of Rpb4 and Rpb7 in yeasts affect the differential gene expression and stress response. Rpb4 is nonessential in S. cerevisiae and affects expression of a small number of genes under normal growth conditions. Here, using a chromatin immunoprecipitation (“ChIP on-chip”) technique, we compared genomewide binding of Rpb4 to that of a core Pol II subunit, Rpb3. Our results showed that in spite of being nonessential for survival, Rpb4 was recruited on coding regions of most transcriptionally active genes, similar to the case with the core Pol II subunit, Rpb3, albeit to a lesser extent. The extent of Rpb4 recruitment increased with increasing gene length. We also observed Pol II lacking Rpb4 to be defective in transcribing long, GC-rich transcription units, suggesting a role for Rpb4 in transcription elongation. This role in transcription elongation was supported by the observed 6-azauracil (6AU) sensitivity of the rpb4Δ mutant. Unlike most phenotypes of rpb4Δ, the 6AU sensitivity of the rpb4Δ strain was not rescued by overexpression of RPB7. This report provides the first instance of a distinct role for Rpb4 in transcription, which is independent of its interacting partner, Rpb7.

RNA isolation from high-phenolic freeze-dried tea (Camellia sinensis) leaves

With minor modifications, we applied a previously reported RNA isolation protocol that used guanidine hydrochloride to leaves of lyophilized (freeze-dried) tea (Camellia sinensis). Plant tissue must be preserved in its collected state, especially when genome-wide expression profiles are studied. Fresh leaf tissues cannot feasibly be transferred at ultra-low temperatures from natural habitats to the laboratory. We explored the use of lyophilized tissue for RNA isolation from tea leaves. High yields of RNA (∼500 μg/g dry weight of leaf tissue) were obtained, and the RNA was suitable for all molecular biology methods tested, including Northern blotting, reverse transcription, and microarray analysis. We demonstrated that RNA obtained from freeze-dried leaf tissue was high quality, undegraded, and useful for all downstream applications.

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 → S transition

The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1→S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1→S arrest is discussed.

The conserved and non-conserved regions of Rpb4 are involved in multiple phenotypes in Saccharomyces cerevisiae.

Rpb4, the fourth largest subunit of RNA polymerase II in Saccharomyces cerevisiae, is required for many phenotypes, including growth at high and low temperatures, sporulation, pseudohyphal growth, activated transcription of a subset of genes, and efficient carbon and energy metabolism. We have used deletion analysis to delineate the domains of the protein involved in these multiple phenotypes. The scRpb4 protein is conserved at the N and C termini but possesses certain non-conserved regions in the central portion. Our deletion analysis and molecular modeling results show that the N- and C-terminal conserved regions of Rpb4 are involved in interaction with Rpb7, the Rpb4 interacting partner in the RNA polymerase II. We further show that the conserved N terminus is required for efficient activated transcription from the INO1 promoter but not the GAL10- or the HSE-containing promoters. The N terminus is not required for any of the stress responses tested: growth at high temperatures, sporulation, and pseudohyphal growth. The conserved C-terminal 23 amino acids are not required for the role of Rpb4 in the pseudohyphal growth phenotype but might play a role in other stress responses and activated transcription. From the deletion analysis of the non-conserved regions, we report that they influence phenotypes involving both the N and C termini (interaction with Rpb7 and transcription from the INO1 promoter) but not any of the stress-responsive phenotypes tested suggesting that they might be involved in maintaining the two conserved domains in an appropriate conformation for interaction with Rpb7 and other proteins. Taken together, our results allow us to assign phenotype-specific roles for the different conserved and non-conserved regions of Rpb4.

Overexpression of the gene for Rpb7 subunit of yeast RNA polymerase II rescues the phenotypes associated with absence of the largest, nonessential subunit Rpb4

The easily dissociable subcomplex of Rpb4 and Rpb7 subunits of yeast RNA polymerase II has been considered, for long, to play a role in stabilizing Pol II under stress. On the basis of previous genetic and biochemical observations, it was proposed that within the subcomplex one of the functions of Rpb4p is to stabilize the interaction between Rpb7p and the rest of Pol II. We took a direct approach to test the latter possibility by overexpression and mutagenesis ofRPB7 in absence of Rpb4p. We report here the results, which support the latter hypothesis. While it has been previously reported that absence of Rpb4p results in reduction in overall transcription by Pol II, our comparative analysis of RNAs fromRPB4 andrpb4δ cells suggests that there are indeed several genes differentially expressed between the two cells. We propose that the qualitative differences in overall transcription in presence and absence of Rpb4p imply a more active role for Rpb4p in transcription of at least a subset of genes.

Basal transcription machinery: role in regulation of stress response in eukaryotes

The holoenzyme of prokaryotic RNA polymerase consists of the core enzyme, made of two α, β, β′ and ω subunits, which lacks promoter selectivity and a sigma (σ) subunit which enables the core enzyme to initiate transcription in a promoter dependent fashion. A stress sigma factor σs, in prokaryotes seems to regulate several stress response genes in conjunction with other stress specific regulators. Since the basic principles of transcription are conserved from simple bacteria to multicellular complex organisms, an obvious question is: what is the identity of a counterpart of σs, that is closest to the core polymerase and that dictates transcription of stress regulated genes in general? In this review, we discuss the logic behind the suggestion that like in prokaryotes, eukaryotes also have a common functional unit in the transcription machinery through which the stress specific transcription factors regulate rapid and highly controlled induction of gene expression associated with generalized stress response and point to some candidates that would fit the bill of the eukaryotic σs.

UDP-glucose 4, 6-dehydratase activity plays an important role in maintaining cell wall integrity and virulence of Candida albicans.

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFNγ and TNFα levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.