Unanchored ubiquitin chains do not lead to marked alterations in gene expression inDrosophila melanogaster

Abstract

The 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.

Unanchored Ubiquitin Chains, Revisited

“…Taken together, these studies (Blount et al, 2018(Blount et al, , 2019(Blount et al, , 2020) add yet more complexity to the general understanding of unanchored poly-Ub. Clearly, free Ub chains are not always toxic.…”

“…We examined changes at the transcriptional level through RNAseq, using flies expressing everywhere Ub 6 -Stop, Ub 6 -GG, or no transgene with the same genetic background. The presence of each type of Ub 6 chain led to significant changes in the expression of approximately 90 fly genes, but with no clear, coordinated cellular response to indicate the induction of any particular pathways (Blount et al, 2019). Only 30% of the altered Drosophila transcripts observed have assigned gene names, and 27% of the genes had no predicted function, opening up the potential for the future identification and characterization of proteins that interact with untethered poly-Ub.…”

“…the low number of altered transcripts and a lack of specificity for Ub-related pathways in those transcripts that were altered (Blount et al, 2019).…”

“…It may seem that more recent studies revealing unanchored Ub's physiological roles and lack of toxicity contradict earlier studies that characterized free poly-Ub as harmful, but the seeming discordance only highlights the importance of Ub chain type and context. Some of the previous in vitro and yeast studies that suggested proteasomal inhibition by untethered poly-Ub focused on Lys48-linked chains (Piotrowski et al, 1997;Thrower et al, 2000;Dayal et al, 2009), while the Ub 6 chains expressed in Drosophila are head-to-tail (Blount et al, 2018(Blount et al, , 2019. In principle, it makes sense that unanchored chains that resemble poly-Ub attached to proteasome-targeted proteinswhich are often Lys48-linked -could outcompete proteasomal substrates in a manner not observed with linear chains that are not traditionally associated with proteasomal degradation (Piotrowski et al, 1997;Thrower et al, 2000;Komander and Rape, 2012).…”

Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-κB Signaling

“…Our previous work introduced transgenic, DUB-resistant, untethered Ub chains into a physiological system, Drosophila melanogaster , to address questions surrounding the potential toxicity and handling of free poly-Ub in an intact, multicellular organism [ 26 , 27 ]. We synthesized DNA to express six Ub molecules in tandem, without any internal “GG” motifs (denoted as Ub 6 ), which prevents them from being cleaved into mono-Ub by DUBs.…”