Abstract
UBC gene plays a critical role in maintaining ubiquitin (Ub) homeostasis. It is upregulated under stress conditions, and herein we report that it is downregulated upon Ub overexpression. Downregulation occurs in a dose-dependent manner, suggesting the existence of a fine-tuned Ub sensing mechanism. This “sensor” requires a conjugation competent ubiquitin to detect Ub levels. Searching the sensor among the transcription factors involved in basal and stress-induced UBC gene expression was unsuccessful. Neither HSF1 and HSF2, nor Sp1 and YY1 are affected by the increased Ub levels. Moreover, mutagenesis of their binding sites in the UBC promoter-driven reporter constructs does not impair the downmodulation effect. Epigenetic studies show that H2A and H2B ubiquitination within the UBC promoter region is unchanged upon ubiquitin overexpression. Noteworthy, quantification of nascent RNA molecules excludes that the downmodulation arises in the transcription initiation step, rather pointing towards a post-transcriptional mechanism. Indeed, a significantly higher fraction of unspliced UBC mRNA is detected in ubiquitin overexpressing cells, compared to empty vector transfected cells. Our findings suggest how increasing cellular ubiquitin levels may control the expression of UBC gene by negatively affecting the splicing of its pre-mRNA, providing a straightforward feedback strategy for the homeostatic control of ubiquitin pools.
Highlights
The protein ubiquitin (Ub) is probably the most important post-translational modifier of the proteome in eukaryotic cells, regulating the stability, function, localization of its target substrates and as such, it controls an array of cellular processes and affects many signaling pathways[1,2]
Downregulation of the UBC gene by exogenous Ub occurred in a dose dependent manner (Fig. 1C), starting from cells transfected with 50 ng of Ub plasmid, where the concentration of ubiquitin was ∼2.4-fold compared to the one detected in pCMV-Myc transfected cells, indicating that this regulatory loop may have a physiological relevance
The same behavior was found for HSP70 mRNA, detected as representative HSF1 target gene (Fig. 4E). These results show that cells respond to MG132 treatment by inducing UBC, despite the high intracellular Ub levels, meaning that the Heat Shock Response (HSR) and the downregulation effect, both impacting on UBC gene expression, are distinct regulatory mechanisms
Summary
The protein ubiquitin (Ub) is probably the most important post-translational modifier of the proteome in eukaryotic cells, regulating the stability, function, localization of its target substrates and as such, it controls an array of cellular processes and affects many signaling pathways[1,2]. When the authors investigated the potential effects of the higher Ub levels on the main components of the ubiquitin-proteasome system, they found a significant decrease in the expression of the endogenous polyubiquitin genes UBC and UBB, arguing that this is consistent with the need for a tightly regulated Ub homeostasis in neurons[25,27] They did not further investigate the molecular mechanisms for this transcriptional downregulation. We found that overexpression of wild-type ubiquitin in different human cell lines (both normal and tumor derived) resulted in lowered levels of UBC and UBB mRNAs; the UBC fold-decrease was directly related to the amount of ubiquitin overexpressed, suggesting that a proper negative feedback regulatory mechanism, able to sense the Ub levels, could act to maintain Ub within a defined concentration range under unstressed conditions Another challenging issue is to highlight the cis-acting elements in the promoter region of UBC and UBB, which make these two genes the main targets and effectors of the ubiquitin sensing mechanism. The harvested data point towards a post-transcriptional ubiquitin-mediated modulation of UBC gene expression
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