Abstract
Ubiquitin is required under both normal and stress conditions. Under stress conditions, upregulation of the polyubiquitin gene UBC is essential to meet the requirement of increased ubiquitin levels to confer stress resistance. However, UBC upregulation is usually observed only under stress conditions and not under normal conditions. Therefore, it has not been possible to upregulate UBC under normal conditions to study the effect of excess ubiquitin on cellular machinery. Recently, the CRISPR/Cas9 system has been widely used in biological research as a useful tool to study gene disruption effects. In this study, using an inducible CRISPR/Cas9 variant, a dCas9–VP64 fusion protein, combined with a single guide RNA (sgRNA) containing MS2 aptamer loops and MS2-p65-HSF1, we developed a system to increase the ubiquitin pool via upregulation of UBC. Although it is challenging to upregulate the expression of a gene that is already expressed at high levels, the significance of our system is that UBC upregulation can be induced in an efficient, reversible manner that is compatible with cellular processes, even under normal conditions. This system can be used to study ubiquitin pool dynamics and it will be a useful tool in identifying the role of ubiquitin under normal and stress conditions.
Highlights
Ubiquitin (Ub) is a highly conserved eukaryotic protein that plays diverse roles within cells [1]
We developed a system to upregulate the polyubiquitin gene UBC, using an inducible CRISPR/Cas9 variant
This system is of particular interest as (1) it can upregulate the stress-responsive gene even under normal conditions; (2) it can upregulate a gene whose expression levels are already high; and (3) its upregulation is dependent on the levels of Cas9 expression and is completely reversible
Summary
Ubiquitin (Ub) is a highly conserved eukaryotic protein that plays diverse roles within cells [1]. The most important role of Ub is to polyubiquitinate its substrates and target them to the proteasome for degradation [2]. Degradation of these substrates occurs under both normal and stress conditions. A well-known antioxidant response pathway protein, Nrf (nuclear factor erythroid 2-related factor 2), is polyubiquitinated and highly prone to degradation under normal conditions, but is stabilized under oxidative stress conditions and translocates into the nucleus to serve as a transcription factor to increase the expression of antioxidant response genes [3,4,5]. Ub plays its role under diverse (normal and stress) conditions to maintain cellular protein homeostasis and its target substrates may differ depending on cellular status
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