Modulation of cyclic nucleotide-mediated cellular signaling and gene expression using photoactivated adenylyl cyclase as an optogenetic tool.

Cyclic nucleotide signaling pathway plays a significant role in various biological processes such as cell growth, transcription, inflammation, in microbial pathogenesis, etc. Modulation of cyclic nucleotide levels by optogenetic tools has overcome certain limitations of studying transduction cascade by pharmacological agents and has allowed several ways to modulate biological processes in a spatiotemporal manner. Here, we have shown the optogenetic modulation of the cyclooxygenase 2 (Cox-2) gene expression and their downstream effector molecule (PGE2) in HEK-293T cells and the development process of Dictyostelium discoideum via modulating the cyclic nucleotide (cAMP) signaling pathway utilizing photoactivated adenylyl cyclases (PACs) as an optogenetic tool. Light-induced activation of PACs in HEK-293T cells increases the cAMP level that leads to activation of cAMP response element-binding protein (CREB) transcription factor and further upregulates downstream Cox-2 gene expression and their downstream effector molecule prostaglandin E2. In D. discoideum, the light-regulated increase in cAMP level affects the starvation-induced developmental process. These PACs could modulate the cAMP levels in a light-dependent manner and have a potential to control gene expression and their downstream effector molecules with varying magnitude. It would enable one to utilize PAC as a tool to decipher cyclic nucleotide mediated signaling pathway regulations and their mechanism.

Functional characterisation of parvulin-type peptidyl prolyl cis-trans isomerase, PinA in Dictyostelium discoideum.

Pin1-type parvulins are unique among PPIases that can catalyse an otherwise slow cis-trans isomerisation of phosphorylated peptide bond preceding proline in target proteins. This prolyl isomerisation process can regulate activity, stability and localisation of target proteins and thus control cellular processes like eukaryotic cell proliferation, cell cycle progression and gene regulation. Towards understanding the function of Pin1-type prolyl isomerisation in Dictyostelium discoideum, a slime mould with distinct growth and developmental phases, we identified PinA as a novel Pin1-type parvulin by its ability to complement the temperature sensitivity phenotype associated with a mutation in ESS1 in S. cerevisiae. In D. discoideum, pinA is temporally and spatially regulated during growth and development. PinA is both nuclear as well as cytoplasmic in the growing cells. We further show that loss of pinA (pinA-) leads to decreased growth rate, reduced spore formation and abnormal prespore-prestalk patterning. We conclude that PinA is required for normal growth as well as development in D. discoideum.

Targeting fungal genes by diced siRNAs: a rapid tool to decipher gene function in Aspergillus nidulans.

BACKGROUND: Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus, Aspergillus nidulans. METHODOLOGY/PRINCIPAL FINDINGS: Stable expression of heterologous reporter gene in A. nidulans eases the examination of a new RNAi-induction route. Hence, we have optimized Agrobacterium tumefaciens-mediated transformation (AMT) of A. nidulans for stable expression of sGFP gene. This study demonstrates that the reporter GFP gene stably introduced into A. nidulans can be effectively silenced by treatment of GFP-d-siRNAs. We have shown the down-regulation of two endogenous genes, AnrasA and AnrasB of A. nidulans by d-siRNAs. We have also elucidated the function of an uncharacterized Ras homolog, rasB gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting AnrasA. Silencing was shown to be sequence-specific, since expression profiles of other closely related Ras family genes in d-siRNA treated AnrasA and AnrasB silenced lines exhibited no change in gene expression. CONCLUSIONS/SIGNIFICANCE: We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in A. nidulans using d-siRNAs. We have also optimized an efficient AMT in A. nidulans, which is useful for stable integration of transgenes.

Identification and characterization of DdRPB4, a subunit of Dictyostelium discoideum RNA polymerase II.

Rpb4, the fourth largest subunit of the eukaryotic RNA polymerase II (RNAPII), is required for growth at extreme temperatures and for an appropriate response to nutrient starvation in yeast. Sequence homologs of Rpb4 are found in most sequenced genomes from yeast to humans. To elucidate the role of this subunit in nutrient starvation, we chose Dictyostelium discoideum, a soil amoeba, which responds to nutrient deprivation by undergoing a complex developmental program. Here we report the identification of homolog of Saccharomyces cerevisiae RPB4 in D. discoideum. Localization and complementation studies suggest that Rpb4 is functionally conserved. DdRPB4 transcript and protein levels are developmentally regulated. Although DdRPB4 could not be deleted, overexpression revealed that the Rpb4 protein is essential for cell survival and is regulated stringently at the post-transcriptional level in D. discoideum. Thus maintaining a critical level of Rpb4 is important for this organism.

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 alpha, beta, beta' and omega subunits, which lacks promoter selectivity and a sigma (sigma) subunit which enables the core enzyme to initiate transcription in a promoter dependent fashion. A stress sigma factor sigma(s), 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 sigma(s), 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 sigma(s).

Relative levels of RNA polII subunits differentially affect starvation response in budding yeast.

The Rpb4/7 subcomplex of RNA polymerase II in Saccharomyces cerevisiae is known to play an important role in stress response and stress survival. These two proteins perform overlapping functions ensuring an appropriate cellular response through transcriptional regulation of gene expression. Rpb4 and Rpb7 also perform many cellular functions either together or independent of one another. Here, we show that Rpb4 and Rpb7 differently affect during the nutritional starvation response pathways of sporulation and pseudohyphae formation. Rpb4 enhances the cells' proficiency to sporulate but suppresses pseudohyphal growth. On the other hand, Rpb7 promotes pseudohyphal growth and suppresses sporulation in a dose-dependent manner. We present a model whereby the stoichiometry of Rpb4 and Rpb7 and their relative levels in the cell play a switch like role in establishing either sporulation or pseudohyphal gene expression.

Domainal organization of the lower eukaryotic homologs of the yeast RNA polymerase II core subunit Rpb7 reflects functional conservation.

The subcomplex of Rpb4 and Rpb7 subunits of RNA pol II in Saccharomyces cerevisiae is known to be an important determinant of transcription under a variety of physiological stresses. In S.cerevisiae, RPB7 is essential for cell viability while rpb4 null strains are temperature sensitive at low and high temperatures. The rpb4 null strain also shows defect in sporulation and a predisposed state of pseudohyphal growth. We show here that, apart from S.cerevisiae Rpb7, the Rpb7 homologs from other lower eukaryotes like Schizosaccharomyces pombe, Candida albicans and Dictyostelium discoideum can complement for the absence of S.cerevisiae RPB7. This is the first report where we have shown that both the C.albicans and D.discoideum homologs are functional orthologs of the yeast RPB7. We also show that high expression levels of S.cerevisiae RPB7 and its homologs rescue the sporulation defect of rpb4 homozygous null diploids, but only some of them cause significant enhancement of the pseudohyphal phenotype. Structural modeling of Rpb7 and its homologs show a high degree of conservation in the overall structure. This study indicates a structural and functional conservation of different Rpb7 across species and also a conserved role of Rpb7 in the subcomplex with respect to nutritional stress.