Stress-sensitive dynamics of miRNAs and Elba1 in Drosophila embryogenesis.

Early-life stress can result in life-long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. Here, we show that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher-order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer-1 mutant, we show that the stress-induced decrease in one of these genes, the insulator-binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV wm4h reporter. We propose that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1.

Dietary Sugar Shifts Mitochondrial Metabolism and Small RNA Biogenesis in Sperm.

Aims: Increasing concentrations of dietary sugar results in a linear accumulation of triglycerides in male Drosophila, while inducing a U-shaped obesity response in their offspring. Here, using a combination of proteomics and small RNA (sRNA) sequencing, we aimed at understanding the molecular underpinning in sperm for such plasticity. Results: Proteomic analysis of seminal vesicles revealed that increasing concentrations of dietary sugar resulted in a bell-shaped induction of proteins involved in metabolic/redox regulation. Using stains and in vivo redox reporter flies, this pattern could be explained by changes in sperm production of reactive oxygen species (ROS), more exactly mitochondria-derived H2O2. By quenching ROS with the antioxidant N-acetyl cysteine and performing sRNA-seq on sperm, we found that sperm miRNA is increased in response to ROS. Moreover, we found sperm mitosRNA to be increased in high-sugar diet conditions (independent of ROS). Reanalyzing our previously published data revealed a similar global upregulation of human sperm mitosRNA in response to a high-sugar diet, suggesting evolutionary conserved mechanisms. Innovation: This work highlights a fast response to dietary sugar in mitochondria-produced H2O2 in Drosophila sperm and identifies redox-sensitive miRNA downstream of this event. Conclusions: Our data support a model where changes in the sperm mitochondria in response to dietary sugar are the primary event, and changes in redox homoeostasis are secondary to mitochondrial ROS production. These data provide multiple candidates for paternal intergenerational metabolic responses as well as potential biomarkers for human male fertility. Antioxid. Redox Signal. 38, 1167-1183.

Asp56 in actin is critical for the full activity of the amino acid starvation-responsive kinase Gcn2.

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.

Domain II of the translation elongation factor eEF1A is required for Gcn2 kinase inhibition.

The signalling pathway governing general control nonderepressible (Gcn)2 kinase allows cells to cope with amino acid shortage. Under starvation, Gcn2 phosphorylates the translation initiation factor eukaryotic translation initiation factor (eIF)2α, triggering downstream events that ultimately allow cells to cope with starvation. Under nutrient-replete conditions, the translation elongation factor eEF1A binds Gcn2 to contribute to keeping Gcn2 inactive. Here, we aimed to map the regions in eEF1A involved in binding and/or regulating Gcn2. We find that eEF1A amino acids 1-221 and 222-315, containing most of domains I and II, respectively, bind Gcn2 in vitro. Overexpression of eEF1A lacking or containing domain III impairs eIF2α phosphorylation. While the latter reduces growth under starvation similarly to eEF1A lacking domain I, the former enhances growth in a Gcn2-dependent manner. Our studies suggest that domain II is required for Gcn2 inhibition and that eEF1A lacking domain III mainly affects the Gcn2 response pathway downstream of Gcn2.

Human sperm displays rapid responses to diet.

The global rise in obesity and steady decline in sperm quality are two alarming trends that have emerged during recent decades. In parallel, evidence from model organisms shows that paternal diet can affect offspring metabolic health in a process involving sperm tRNA-derived small RNA (tsRNA). Here, we report that human sperm are acutely sensitive to nutrient flux, both in terms of sperm motility and changes in sperm tsRNA. Over the course of a 2-week diet intervention, in which we first introduced a healthy diet followed by a diet rich in sugar, sperm motility increased and stabilized at high levels. Small RNA-seq on repeatedly sampled sperm from the same individuals revealed that tsRNAs were up-regulated by eating a high-sugar diet for just 1 week. Unsupervised clustering identified two independent pathways for the biogenesis of these tsRNAs: one involving a novel class of fragments with specific cleavage in the T-loop of mature nuclear tRNAs and the other exclusively involving mitochondrial tsRNAs. Mitochondrial involvement was further supported by a similar up-regulation of mitochondrial rRNA-derived small RNA (rsRNA). Notably, the changes in sugar-sensitive tsRNA were positively associated with simultaneous changes in sperm motility and negatively associated with obesity in an independent clinical cohort. This rapid response to a dietary intervention on tsRNA in human sperm is attuned with the paternal intergenerational metabolic responses found in model organisms. More importantly, our findings suggest shared diet-sensitive mechanisms between sperm motility and the biogenesis of tsRNA, which provide novel insights about the interplay between nutrition and male reproductive health.

Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2α.

The common method for liberating proteins from Saccharomyces cerevisiae cells involves mechanical cell disruption using glass beads and buffer containing inhibitors (protease, phosphatase and/or kinase inhibitors), followed by centrifugation to remove cell debris. This procedure requires the use of costly inhibitors and is laborious, in particular when many samples need to be processed. Also, enzymatic reactions can still occur during harvesting and cell breakage. As a result low-abundance and labile proteins may be degraded, and enzymes such as kinases and phosphatases may still modify proteins during and after cell lysis. We believe that our rapid sample preparation method helps overcome the above issues and offers the following advantages: (a) it is cost-effective, as no inhibitors and breaking buffer are needed; (b) cell breakage is fast (about 15 min) since it only involves a few steps; (c) the use of formaldehyde inactivates endogenous proteases prior to cell lysis, dramatically reducing the risk of protein degradation; (d) centrifugation steps only occur prior to cell lysis, circumventing the problem of losing protein complexes, in particular if cells were treated with formaldehyde intended to stabilize and capture large protein complexes; and (e) since formaldehyde has the potential to instantly terminate protein activity, this method also allows the study of enzymes in live cells, i.e. in their true physiological environment, such as the short-term effect of a drug on enzyme activity. Taken together, the rapid sample preparation procedure provides a more accurate snapshot of the cell's protein content at the time of harvesting. Copyright © 2017 John Wiley & Sons, Ltd.

Keeping the eIF2 alpha kinase Gcn2 in check.

The protein kinase Gcn2 is present in virtually all eukaryotes and is of increasing interest due to its involvement in a large array of crucial biological processes. Some of these are universally conserved from yeast to humans, such as coping with nutrient starvation and oxidative stress. In mammals, Gcn2 is important for e.g. long-term memory formation, feeding behaviour and immune system regulation. Gcn2 has been also implicated in diseases such as cancer and Alzheimer's disease. Studies on Gcn2 have been conducted most extensively in Saccharomyces cerevisiae, where the mechanism of its activation by amino acid starvation has been revealed in most detail. Uncharged tRNAs stimulate Gcn2 which subsequently phosphorylates its substrate, eIF2α, leading to reduced global protein synthesis and simultaneously to increased translation of specific mRNAs, e.g. those coding for Gcn4 in yeast and ATF4 in mammals. Both proteins are transcription factors that regulate the expression of a myriad of genes, thereby enabling the cell to initiate a survival response to the initial activating cue. Given that Gcn2 participates in many diverse processes, Gcn2 itself must be tightly controlled. Indeed, Gcn2 is regulated by a vast network of proteins and RNAs, the list of which is still growing. Deciphering molecular mechanisms underlying Gcn2 regulation by effectors and inhibitors is fundamental for understanding how the cell keeps Gcn2 in check ensuring normal organismal function, and how Gcn2-associated diseases may develop or may be treated. This review provides a critical evaluation of the current knowledge on mechanisms controlling Gcn2 activation or activity.

Semi-quantitative colony immunoassay for determining and optimizing protein expression in Saccharomyces cerevisiae and Escherichia coli.

This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl ß-D-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.