Abstract
ABSTRACTThe small protein modifier ubiquitin regulates various aspects of cellular biology through its chemical conjugation onto proteins. Ubiquitination of proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Ubiquitin chains can be attached onto other proteins or can exist as unanchored species, i.e. free from another protein. Unanchored ubiquitin chains are thought to be deleterious to the cell and rapidly disassembled into mono-ubiquitin. We recently examined the toxicity and utilization of unanchored poly-ubiquitin in Drosophila melanogaster. We found that free poly-ubiquitin species are largely innocuous to flies and that free poly-ubiquitin can be controlled by being degraded by the proteasome or by being conjugated onto another protein as a single unit. Here, to explore whether an organismal defense is mounted against unanchored chains, we conducted RNA-Seq analyses to examine the transcriptomic impact of free poly-ubiquitin in the fly. We found ∼90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was essentially devoid of ubiquitin-, proteasome-, or autophagy-related components. The seeming absence of a large and multipronged response to unanchored poly-ubiquitin supports the conclusion that these species need not be toxic in vivo and underscores the need to re-examine the role of free ubiquitin chains in the cell.
Highlights
Cellular and organismal physiology and homeostasis are regulated at multiple, inter-dependent levels that extend from DNA-based regulation of gene expression to the epigenetic control of genes themselves and of their products
Transgenes with upstream activating sequence (UAS) sites are activated under the control of the transcription factor Gal4, itself expressed in the pattern of a specific gene (Brand and Perrimon, 1993; Brand et al, 1994)
For our work in this study, we selected the Gal4 driver sqh-Gal4 (Kiehart et al, 2004; Franke et al, 2006, 2010; Todi et al, 2005, 2008) to express either form of Ub6 in all fly tissues, throughout development and in adults. This driver employs the promoter and expression pattern of the gene spaghetti squash, which encodes the regulatory light chain of non-muscle type 2 myosin. sqh-Gal4 is a strong driver that leads to high levels of UAS-based transgene expression, during all developmental stages and throughout adulthood
Summary
Cellular and organismal physiology and homeostasis are regulated at multiple, inter-dependent levels that extend from DNA-based regulation of gene expression to the epigenetic control of genes themselves and of their products. Ubiquitin (Ub), itself a small protein of approximately 8.5 kDa, is highly conserved among all eukaryotic species and regulates proteins in various ways, from tagging them for proteasomal degradation to directing their participation in cellular signaling pathways (Komander and Rape, 2012; Ristic et al, 2014; Asaoka et al, 2016). Ub conjugation onto another protein – what is termed ubiquitination – requires the concerted effort of three types of enzymes: an E1 activating enzyme, an E2 conjugating enzyme and an E3 ligase (Fig. 1A). Ubiquitination is a reversible process; deubiquitinases (DUBs) remove Ub from a protein or edit the length and type of a Ub chain (Fig. 1A; Swatek and Komander, 2016)
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