“Sleeping Beauty”: Quiescence in Saccharomyces cerevisiae

The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one “model” quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.     

CDT1 is the major functional regulatory subunit of the pre‐replication complex in zygotes

Pre‐replication complex (pre‐RC) is critical for DNA replication initiation. CDT1 and MCM2 are the subunits of pre‐RC, and proper regulation of CDT1 and MCM2 are necessary for DNA replication and cell proliferation. The present study aimed to explore the role of CDT1 and MCM2 in oocyte meiotic maturation and early embryonic development. The depletion and overexpression of Cdt1 and Mcm2 in oocyte and zygote were achieved by microinjecting specific siRNA and mRNA to explored their functions in oocyte meiotic maturation and embryonic development. Then, we examined the effect of CDT1 and MCM2 on other signal pathways by immunostaining the expression of related maker genes. We showed that neither depletion nor overexpression of Cdt1 affected oocyte meiotic progressions. The CDT1 was degraded in S phase and remained at a low level in G2 phase of zygote. Exogenous expression of Cdt1 in G2 phase led to embryo attest at zygote stage. Mechanistically, CDT1 overexpression induced DNA re‐replication and thus DNA damage check‐point activation. Protein abundance of MCM2 was stable throughout the cell cycle, and embryos with overexpressed MCM2 could develop to blastocysts normally. Overexpression or depletion of Mcm2 also had no effect on oocyte meiotic maturation. Our results indicate that pre‐RC subunits CDT1 and MCM2 are not involved in oocyte meiotic maturation. In zygote, CDT1 but not MCM2 is the major regulator of DNA replication in a cell cycle dependent manner. Furthermore, its'' degradation is essential for zygotes to prevent from DNA re‐replication in G2 stage.

Pre‐replication complex (pre‐RC) is formed in G1 phase during which CDT1 is localized in nucleus. When zygote enters S phase, CDT1 is degraded and stays at a low level in G2 phase. However, the expression of exogenous Cdt1 mRNA in G2 phase of fertilized egg, mimicking events in which CDT1 degradation is disrupted, pre‐RC reassembles and leads to DNA re‐replication, and thus DNA damage check point activation, which results in embryo arrest at G2/M phase.     

Ribosome biogenesis is sensed at the Start cell cycle checkpoint

In the yeast Saccharomyces cerevisiae it has long been thought that cells must reach a critical cell size, called the "setpoint," in order to allow the Start cell cycle transition. Recent evidence suggests that this setpoint is lowered when ribosome biogenesis is slowed. Here we present evidence that yeast can sense ribosome biogenesis independently of mature ribosome levels and protein synthetic capacity. Our results suggest that ribosome biogenesis directly promotes passage through Start through Whi5, the yeast functional equivalent to the human tumor suppressor Rb. When ribosome biogenesis is inhibited, a Whi5-dependent mechanism inhibits passage through Start before significant decreases in both the number of ribosomes and in overall translation capacity of the cell become evident. This delay at Start in response to decreases in ribosome biogenesis occurs independently of Cln3, the major known Whi5 antagonist. Thus ribosome biogenesis may be sensed at multiple steps in Start regulation. Ribosome biogenesis may thus both delay Start by increasing the cell size setpoint and independently may promote Start by inactivating Whi5.     

Nucleolar c‐Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood‐borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein–Barr virus nuclear antigen 1‐binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpA^L10A) did not alter its translocation to the nucleus but abolished the effector’s capacity to interact with EBP2. VgpA‐EBP2 interaction led to the re‐localization of c‐Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA‐EBP2 interaction elevated EBP2’s affinity for c‐Myc and prolonged the oncoprotein’s half‐life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re‐localization of c‐Myc. Moreover, the in vivo VgpA‐EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus’ colonization and virulence. These observations suggest that direct effector stimulation of a c‐Myc controlled host cell growth program can contribute to pathogenesis.     

“Start” Mutants of Saccharomyces cerevisiae Are Suppressed in Carbon Catabolite-Derepressing Medium

Temperature-sensitive cell division “start” mutants cdc28, cdc36, cdc37, and cdc39 of the yeast Saccharomyces cerevisiae arrested cell division in the G1 phase of the cell cycle in glucose medium. I report here that cdc28, cdc36, and cdc39 mutants were suppressed when grown in carbon catabolite-derepressing medium.     

“Protein aggregates” contain RNA and DNA,entrapped by misfolded proteins but largely rescued by slowing translational elongation

All neurodegenerative diseases feature aggregates, which usually contain disease‐specific diagnostic proteins; non‐protein constituents, however, have rarely been explored. Aggregates from SY5Y‐APP_Sw neuroblastoma, a cell model of familial Alzheimer''s disease, were crosslinked and sequences of linked peptides identified. We constructed a normalized “contactome” comprising 11 subnetworks, centered on 24 high‐connectivity hubs. Remarkably, all 24 are nucleic acid‐binding proteins. This led us to isolate and sequence RNA and DNA from Alzheimer''s and control aggregates. RNA fragments were mapped to the human genome by RNA‐seq and DNA by ChIP‐seq. Nearly all aggregate RNA sequences mapped to specific genes, whereas DNA fragments were predominantly intergenic. These nucleic acid mappings are all significantly nonrandom, making an artifactual origin extremely unlikely. RNA (mostly cytoplasmic) exceeded DNA (chiefly nuclear) by twofold to fivefold. RNA fragments recovered from AD tissue were ~1.5‐to 2.5‐fold more abundant than those recovered from control tissue, similar to the increase in protein. Aggregate abundances of specific RNA sequences were strikingly differential between cultured SY5Y‐APP_Sw glioblastoma cells expressing APOE3 vs. APOE4, consistent with APOE4 competition for E‐box/CLEAR motifs. We identified many G‐quadruplex and viral sequences within RNA and DNA of aggregates, suggesting that sequestration of viral genomes may have driven the evolution of disordered nucleic acid‐binding proteins. After RNA‐interference knockdown of the translational‐procession factor EEF2 to suppress translation in SY5Y‐APP_Sw cells, the RNA content of aggregates declined by >90%, while reducing protein content by only 30% and altering DNA content by ≤10%. This implies that cotranslational misfolding of nascent proteins may ensnare polysomes into aggregates, accounting for most of their RNA content.     

Putting the brakes on phagocytosis: “don't‐eat‐me” signaling in physiology and disease

Timely removal of dying or pathogenic cells by phagocytes is essential to maintaining host homeostasis. Phagocytes execute the clearance process with high fidelity while sparing healthy neighboring cells, and this process is at least partially regulated by the balance of “eat‐me” and “don''t‐eat‐me” signals expressed on the surface of host cells. Upon contact, eat‐me signals activate “pro‐phagocytic” receptors expressed on the phagocyte membrane and signal to promote phagocytosis. Conversely, don''t‐eat‐me signals engage “anti‐phagocytic” receptors to suppress phagocytosis. We review the current knowledge of don''t‐eat‐me signaling in normal physiology and disease contexts where aberrant don''t‐eat‐me signaling contributes to pathology.     

Hypophosphorylated pRb knock‐in mice exhibit hallmarks of aging and vitamin C‐preventable diabetes

Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb^∆K4 and Rb^∆K7 knock‐in mice, in which either four or all seven phosphorylation sites in the C‐terminal region of pRb, respectively, have been abolished by Ser/Thr‐to‐Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb^∆K4 mice exhibit telomere attrition but no other abnormalities, Rb^∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb^∆K7 mice is insulin‐sensitive and associated with failure of quiescent pancreatic β‐cells to re‐enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence‐associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre‐treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re‐entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK‐inhibitor therapeutics, diabetes, and longevity.     

An assessment of terminology for intraspecific diversity in fishes,with a focus on “ecotypes” and “life histories”

Understanding and preserving intraspecific diversity (ISD) is important for species conservation. However, ISD units do not have taxonomic standards and are not universally recognized. The terminology used to describe ISD is varied and often used ambiguously. We compared definitions of terms used to describe ISD with use in recent studies of three fish taxa: sticklebacks (Gasterosteidae), Pacific salmon and trout (Oncorhynchus spp., “PST”), and lampreys (Petromyzontiformes). Life history describes the phenotypic responses of organisms to environments and includes biological parameters that affect population growth or decline. Life‐history pathway(s) are the result of different organismal routes of development that can result in different life histories. These terms can be used to describe recognizable life‐history traits. Life history is generally used in organismal‐ and ecology‐based journals. The terms paired species/species pairs have been used to describe two different phenotypes, whereas in some species and situations a continuum of phenotypes may be expressed. Our review revealed overlapping definitions for race and subspecies, and subspecies and ecotypes. Ecotypes are genotypic adaptations to particular environments, and this term is often used in genetic‐ and evolution‐based journals. “Satellite species” is used for situations in which a parasitic lamprey yields two or more derived, nonparasitic lamprey species. Designatable Units, Evolutionary Significant Units (ESUs), and Distinct Population Segments (DPS) are used by some governments to classify ISD of vertebrate species within distinct and evolutionary significant criteria. In situations where the genetic or life‐history components of ISD are not well understood, a conservative approach would be to call them phenotypes.

The terminology used to describe intraspecific diversity is varied and often used ambiguously. “Ecotype” was originally used to describe patterns in genes and ecology, and recent studies employing this term tend to report a genetic basis in ISD. By contrast, “life history” describes biological parameters that affect demography, and this term tends to be used in organismal‐ and ecology‐based journals.     

NHP2 downregulation counteracts hTR‐mediated activation of the DNA damage response at ALT telomeres

About 10% of cancer cells employ the “alternative lengthening of telomeres” (ALT) pathway instead of re‐activating the hTERT subunit of human telomerase. The hTR RNA subunit is also abnormally silenced in some ALT⁺ cells not expressing hTERT, suggesting a possible negative non‐canonical impact of hTR on ALT. Indeed, we show that ectopically expressed hTR reduces phosphorylation of ssDNA‐binding protein RPA (p‐RPA^S33) at ALT telomeres by promoting the hnRNPA1‐ and DNA‐PK‐dependent depletion of RPA. The resulting defective ATR checkpoint signaling at telomeres impairs recruitment of the homologous recombination protein, RAD51. This induces ALT telomere fragility, increases POLD3‐dependent C‐circle production, and promotes the recruitment of the DNA damage marker 53BP1. In ALT⁺ cells that naturally retain hTR expression, NHP2 H/ACA ribonucleoprotein levels are downregulated, likely in order to restrain DNA damage response (DDR) activation at telomeres through reduced 53BP1 recruitment. This unexpected role of NHP2 is independent from hTR’s non‐canonical function in modulating telomeric p‐RPA^S33. Collectively, our study shines new light on the interference between telomerase‐ and ALT‐dependent pathways and unravels a crucial role for hTR and NHP2 in DDR regulation at ALT telomeres.     

Cancer‐secreted miRNAs regulate amino‐acid‐induced mTORC1 signaling and fibroblast protein synthesis

Metabolic reprogramming of non‐cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer‐derived metabolic and non‐metabolic factors, is an emerging aspect of cancer–host interaction. We show that in normal and cancer‐associated fibroblasts, breast cancer‐secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer‐derived miR‐105 and miR‐204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re‐feeding, ¹³C‐arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer‐derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR‐105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re‐prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma‐produced proteins during periodic nutrient fluctuations.     

IL‐21 has a critical role in establishing germinal centers by amplifying early B cell proliferation

The proliferation and differentiation of antigen‐specific B cells, including the generation of germinal centers (GC), are prerequisites for long‐lasting, antibody‐mediated immune protection. Affinity for antigen determines B cell recruitment, proliferation, differentiation, and competitiveness in the response, largely through determining access to T cell help. However, how T cell‐derived signals contribute to these outcomes is incompletely understood. Here, we report how the signature cytokine of follicular helper T cells, IL‐21, acts as a key regulator of the initial B cell response by accelerating cell cycle progression and the rate of cycle entry, increasing their contribution to the ensuing GC. This effect occurs over a wide range of initial B cell receptor affinities and correlates with elevated AKT and S6 phosphorylation. Moreover, the resultant increased proliferation can explain the IL‐21‐mediated promotion of plasma cell differentiation. Collectively, our data establish that IL‐21 acts from the outset of a T cell‐dependent immune response to increase cell cycle progression and fuel cyclic re‐entry of B cells, thereby regulating the initial GC size and early plasma cell output.