Current Genetics最新文献

The resource intensive process of accurate ribosome synthesis is essential for cell viability in all organisms. Ribosome synthesis regulation centers on RNA polymerase I (pol I) transcription of a 35S rRNA precursor that is processed into the mature 18S, 5.8S and 25S rRNAs. During nutrient deprivation or stress, pol I synthesis of rRNA is dramatically reduced. Conversely, chronic stress such as mitochondrial dysfunction induces RNA polymerase II (pol II) to transcribe functional rRNA using an evolutionarily conserved cryptic pol II rDNA promoter suggesting a universal phenomenon. However, this polymerase switches and its role in regulation of rRNA synthesis remain unclear. In this paper, we demonstrate that extended nitrogen deprivation induces the polymerase switch via components of the environmental stress response. We further show that the switch is repressed by Sch9 and activated by the stress kinase Rim15. Like stress-induced genes, the switch requires not only pol II transcription machinery, including the mediator, but also requires the HDAC, Rpd3 and stress transcription factor Hsf1. The current work shows that the constitutive allele, Hsf1^PO4* displays elevated levels of induction in non-stress conditions while binding to a conserved site in the pol II rDNA promoter upstream of the pol I promoter. Whether the polymerase switch serves to provide rRNA when pol I transcription is inhibited or fine-tunes pol I initiation via RNA interactions is yet to be determined. Identifying the underlying mechanism for this evolutionary conserved phenomenon will help understand the mechanism of pol II rRNA synthesis and its role in stress adaptation.

Hydrogen peroxide (H₂O₂) is a debriding agent that damages the microbial structure and function by generating various reactive oxygen species (ROS). H₂O₂-produced hydroxyl radical (OH∙) also exerts oxidative stress on microorganisms. The spread of antibiotic-resistance in bacteria is a serious issue worldwide, and greater efforts are needed to identify and characterize novel antibacterial mechanisms to develop new treatment strategies. Therefore, this study aimed to clarify the relationship between H₂O₂ and Escherichia coli and to elucidate a novel antibacterial mechanism(s) of H₂O₂. Following H₂O₂ exposure, increased levels of 8-hydroxydeoxyguanosine and malondialdehyde indicated that H₂O₂ accelerates oxidation of bacterial DNA and lipids in E. coli. As oxidative damage worsened, the SOS response was triggered. Cell division arrest and resulting filamentous cells were identified in cells, indicating that LexA was involved in DNA replication. It was also verified that RecA, a representative SOS gene, helps self-cleavage of LexA and acts as a bacterial caspase-like protein. Our findings also showed that dinF is essential to preserve E. coli from H₂O₂-induced ROS, and furthermore, demonstrated that H₂O₂-induced SOS response and SOS genes participate differently in guarding E. coli from oxidative stress. As an extreme SOS response is considered apoptosis-like death (ALD) in bacteria, additional experiments were performed to examine the characteristics of ALD. DNA fragmentation and membrane depolarization appeared in H₂O₂-treated cells, suggesting that H₂O₂ causes ALD in E. coli. In conclusion, our investigations revealed that ALD is a novel antibacterial mode of action(s) of H₂O₂ with important contributions from SOS genes.

Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast, Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80 bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80 bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80 bp or 1 kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 in N. liquefaciens conferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.

Temperature fluctuation is one of the most frequent threats to which organisms are exposed in nature. The activation of gene expression programs that trigger the transcription of heat stress-protective genes is the main cellular response to resist high temperatures. In addition, reversible accumulation and compartmentalization of thermosensitive proteins in high-order molecular assemblies are emerging as critical mechanisms to ensure cellular protection upon heat stress. Here, we summarize representative examples of membrane-less intracellular bodies formed upon heat stress in yeasts and human cells and highlight how protein aggregation can be turned into a cytoprotective mechanism.

Stress granule (SG) assembly is a conserved cellular strategy that copes with stress-related damage and promotes cell survival. SGs form through a process of liquid-liquid phase separation. Cellular signaling also appears to employ SG assembly as a mechanism for controlling cell survival and cell death by spatial compartmentalization of signal-transducing factors. While several lines of evidence highlight the importance of SGs as signaling hubs, where protein components of signaling pathways can be temporarily sequestered, shielded from the cytoplasm, the regulation and physiological significance of SGs in this aspect remain largely obscure. A recent study of the heat-shock response in the fission yeast Schizosaaccharomyces pombe provides an unexpected answer to this question. Recently, we demonstrated that the PKC orthologue Pck2 in fission yeast translocates into SGs through phase separation in a PKC kinase activity-dependent manner upon high-heat stress (HHS). Importantly, the downstream MAPK Pmk1 promotes Pck2 recruitment into SGs, which intercepts MAPK hyperactivation and cell death, thus posing SGs as a negative feedback circuit in controlling MAPK signaling. Intriguingly, HHS, but not modest-heat stress targets Pck2 to SGs, independent of canonical SG machinery. Finally, cells fail to activate MAPK signaling when Pck2 is sequestrated into SGs. In this review, we will discuss how SGs have a role as signaling hubs beyond serving as a repository for non-translated mRNAs during acute stress.

Cellular systems depend on multiprotein complexes whose functionalities require defined stoichiometries of subunit proteins. Proper stoichiometry is achieved by controlling the amount of protein synthesis and degradation even in the presence of genetic perturbations caused by changes in gene dosage. As a consequence of increased gene copy number, excess subunits unassembled into the complex are synthesized and rapidly degraded by the ubiquitin-proteasome system. This mechanism, called protein-level dosage compensation, is widely observed not only under such perturbed conditions but also in unperturbed physiological cells. Recent studies have shown that recognition of unassembled subunits and their selective degradation are intricately regulated. This review summarizes the nature, strategies, and increasing complexity of protein-level dosage compensation and discusses possible mechanisms for controlling proteome stoichiometry in multiple layers of biological processes.

The yeast prions (infectious proteins) [URE3] and [PSI+] are essentially non-functional (or even toxic) amyloid forms of Ure2p and Sup35p, whose normal function is in nitrogen catabolite repression and translation termination, respectively. Yeast has an array of systems working in normal cells that largely block infection with prions, block most prion formation, cure most nascent prions and mitigate the toxic effects of those prions that escape the first three types of systems. Here we review recent progress in defining these anti-prion systems, how they work and how they are regulated. Polymorphisms of the prion domains partially block infection with prions. Ribosome-associated chaperones ensure proper folding of nascent proteins, thus reducing [PSI+] prion formation and curing many [PSI+] variants that do form. Btn2p is a sequestering protein which gathers [URE3] amyloid filaments to one place in the cells so that the prion is often lost by progeny cells. Proteasome impairment produces massive overexpression of Btn2p and paralog Cur1p, resulting in [URE3] curing. Inversely, increased proteasome activity, by derepression of proteasome component gene transcription or by 60S ribosomal subunit gene mutation, prevents prion curing by Btn2p or Cur1p. The nonsense-mediated decay proteins (Upf1,2,3) cure many nascent [PSI+] variants by associating with Sup35p directly. Normal levels of the disaggregating chaperone Hsp104 can also cure many [PSI+] prion variants. By keeping the cellular levels of certain inositol polyphosphates / pyrophosphates low, Siw14p cures certain [PSI+] variants. It is hoped that exploration of the yeast innate immunity to prions will lead to discovery of similar systems in humans.

A major question in cell biology is, how are organelles and macromolecular machines moved within a cell? The delivery of cargoes to the right place at the right time within a cell is critical to cellular health. Failure to do so is often catastrophic for animal physiology and results in diseases of the gut, brain, and skin. In budding yeast, a myosin V motor, Myo2, moves cellular materials from the mother cell into the growing daughter bud. Myo2-based transport ensures that cellular contents are shared during cell division. During transport, Myo2 is often linked to its cargo via cargo-specific adaptor proteins. This simple organism thus serves as a powerful tool to study how myosin V moves cargo, such as organelles. Some critical questions include how myosin V moves along the actin cytoskeleton, or how myosin V attaches to cargo in the mother. Other critical questions include how the cargo is released from myosin V when it reaches its final destination in the bud. Here, we review the mechanisms that regulate the vacuole-specific adaptor protein, Vac17, to ensure that Myo2 delivers the vacuole to the bud and releases it at the right place and the right time. Recent studies have revealed that Vac17 is regulated by ubiquitylation and phosphorylation events that coordinate its degradation and the detachment of the vacuole from Myo2. Thus, multiple post-translational modifications tightly coordinate cargo delivery with cellular events. It is tempting to speculate that similar mechanisms regulate other cargoes and molecular motors.

Multicellularity has evolved numerous times across the tree of life. One of the most fundamental distinctions among multicellular organisms is their developmental mode: whether they stay together during growth and develop clonally, or form a group through the aggregation of free-living cells. The five eukaryotic lineages to independently evolve complex multicellularity (animals, plants, red algae, brown algae, and fungi) all develop clonally. This fact has largely been explained through social evolutionary theory's lens of cooperation and conflict, where cheating within non-clonal groups has the potential to undermine multicellular adaptation. Multicellular organisms that form groups via aggregation could mitigate the costs of cheating by evolving kin recognition systems that prevent the formation of chimeric groups. However, recent work suggests that selection for the ability to aggregate quickly may constrain the evolution of highly specific kin recognition, sowing the seeds for persistent evolutionary conflict. Importantly, other features of aggregative multicellular life cycles may independently act to constrain the evolution of complex multicellularity. All known aggregative multicellular organisms are facultatively multicellular (as opposed to obligately multicellular), allowing unicellular-level adaptation to environmental selection. Because they primarily exist in a unicellular state, it may be difficult for aggregative multicellular organisms to evolve multicellular traits that carry pleiotropic cell-level fitness costs. Thus, even in the absence of social conflict, aggregative multicellular organisms may have limited potential for the evolution of complex multicellularity.

Fungal pathogens, from phytopathogenic fungus to human pathogens, are able to alternate between the yeast-like form and filamentous forms. This morphological transition (dimorphism) is in close connection with their pathogenic lifestyles and with their responses to changing environmental conditions. The mechanisms governing these morphogenetic conversions are still not fully understood. Therefore, we studied the filamentous growth of the less-known, non-pathogenic dimorphic fission yeast, S. japonicus, which belongs to an ancient and early evolved branch of the Ascomycota. Its RNA sequencing revealed that several hundred genes were up- or down-regulated in the hyphae compared to the yeast-phase cells. These genes belonged to different GO categories, confirming that mycelial growth is a rather complex process. The genes of transport- and metabolic processes appeared especially in high numbers among them. High expression of genes involved in glycolysis and ethanol production was found in the hyphae, while other results pointed to the regulatory role of the protein kinase A (PKA) pathway. The homologues of 49 S. japonicus filament-associated genes were found by sequence alignments also in seven distantly related dimorphic and filamentous species. The comparative genomic analyses between S. japonicus and the closely related but non-dimorphic S. pombe shed some light on the differences in their genomes. All these data can contribute to a better understanding of hyphal growth and those genomic rearrangements that underlie it.