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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., Funding Agencies|Swedish Research Council [2015-03141]; Ragnar Soderbergs foundation; Knut and Alice Wallenberg foundation [2015.0165]

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)2 alpha, 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 eIF2 alpha 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., Funding Agencies|Marsden Fund CouncilRoyal Society of New ZealandMarsden Fund (NZ) [MAU0607]; Auckland Medical Research Foundation [4113010]; Massey University Strategic Research Excellence Fund [RM20783]; Massey University Doctoral Scholarship; School of Natural and Computational Sciences

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., Funding Agencies|Swedish Research CouncilSwedish Research Council [201503141]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation [2015.0165]; Strategic Research Area Health Care Science at Karolinska Institutet/Umea University; Ragnar Soderberg

Eukaryotic cells regulate protein synthesis (translation) for a rapid response to various types of stress, and this involves several protein-protein interactions (PPIs) and protein phosphorylation. Phosphorylation of eukaryotic initiation factor-2 α (eIF2α) is a common regulatory mechanism to adjust protein synthesis in response to various stimuli. Gcn2 (General Control Non-derepressible) is an eIF2α kinase that is conserved from yeast to mammals, that is activated in response to amino acid starvation. Gcn2 activation leads to a reduction in global protein synthesis and simultaneous augmented translation of GCN4, a transcriptional activator of genes that are necessary to overcome stress. This cascade of events that allows cells in stress adaptation constitutes the General Amino Acid control (GAAC) pathway in yeast. Gcn2 activity is controlled by a large array of proteins that directly or indirectly regulate Gcn2. Gcn2 has to bind another protein called Gcn1, in order to be activated in response to amino acid starvation. Yih1 (Yeast IMPACT homolog 1) in yeast and its counterpart IMPACT (IMPrinted and AnCienT) in mammals are homologous proteins that indirectly regulate Gcn2. Yih1/IMPACT inhibit Gcn2 by competing for Gcn1 binding. Yih1 associates with Actin, and studies so far have suggested that Yih1 only inhibits Gcn2 when it dissociates from Actin. The focus of this thesis work was to shed more light on those interactions relevant for Gcn2 regulation. Firstly, we have identified that the Yih1 mediated interactions occur at distinct cellular locations within the cell, supporting the idea that spatially restricted cellular interactions controlled Gcn2 function. Using in vitro studies we have identified the regions on eEF1A that are involved in Gcn2 and Yih1 binding. The distinct binding sites for both proteins on eEF1A led to further investigations on how the dynamics of these interactions involving eEF1A might affect Gcn2 function. Together with unpublished observati