Dynamic transcription of ubiquitin genes under basal and stressful conditions and new insights into the multiple UBC transcript variants

Highlights

• Polyubiquitin genes UBC and UBB mainly contribute to Ub content in basal conditions.

• UBC is the most responsive gene to differently induced proteotoxic stress.

• Ub ribosomal fusion genes are not involved in the proteotoxic stress response.

• UV-induced genotoxic stress acts post-transcriptionally by stabilizing Ub mRNAs.

• Multiple transcription start sites were identified within the UBC promoter.

Abstract

Ubiquitin (Ub) is a small 76-amino acid protein that is engaged in many different pathways within the cell, including protein turnover. During proteotoxic stress, when the demand of clearing damaged/misfolded proteins strongly increases, cells activate Ub gene transcription to face the need of extra ubiquitin. This paper shows the contribution of the four ubiquitin coding genes (UBB, UBC, UBA52, RPS27A) to the ubiquitin RNA pool under basal and stressful conditions. Our results reveal that UBC and RPS27A represent the major fraction of the Ub transcriptome in different cell lines, but when converted to the coding potential, polyubiquitin genes UBC and UBB mainly contribute to determine the intracellular ubiquitin content under basal conditions. Both the polyubiquitin genes UBB and UBC are upregulated upon proteasome inhibition and oxidative stress, with markedly higher responses from the UBC promoter. A similar output, with lower fold-inductions, is detected in heat-stressed cells, with UBC acting as the main contributor to thermotolerance. By contrast, upon these stressors, the levels of UBA52 and RPS27A mRNAs remain unchanged. Remarkably, UV irradiation fails to induce Ub gene transcription, but rather seems to act at the post-transcriptional level, by stabilizing ubiquitin mRNAs at UV doses which induce rapid degradation of other RNA molecules. Moreover, the evidence that the UBC core promoter contains multiple transcription start sites and their responsiveness to stress, is here reported for the first time.

Introduction

Ubiquitin (Ub) is a versatile 76 amino acid protein that plays important roles inside cells. First characterized for tagging proteins to degradation by the proteasome (Finley, 2009), it is now known to be also involved in processes as varied as signal transduction, endocytosis, transcription and DNA repair (Chen and Sun, 2009). To perform its functions, Ub is linked to target substrates through an isopeptide linkage formed by a three-step enzymatic reaction: activation–conjugation–ligation, catalyzed by E1, E2 and E3 enzymes, respectively (Hershko and Ciechanover, 1998). This post-translational modification can lead to a monoubiquitination or, reiterating the conjugation process with ubiquitin internal lysines, polyubiquitination of the target substrates. Therefore, ubiquitin exists inside cells as free Ub (readily available for ubiquitination), conjugated Ub (covalently attached to substrates) and free unanchored Ub chains. These forms are in dynamic equilibrium, since conjugation and de-conjugation are continuously ongoing processes.

Like other post-translational modifications, ubiquitination is indeed reversible. A class of enzymes is in charge of this process: deubiquitinating enzymes (DUBs) recycle ubiquitin chains in order to maintain free ubiquitin pool (Nijman et al., 2005, Reyes-Turcu et al., 2009).

Although ubiquitin is an abundant protein, representing up to 5% of total proteins, the pool of free unconjugated ubiquitin is surprisingly small: this means that, despite its pervasive roles in many cell functions, Ub is not produced in excess. Free ubiquitin levels in a cell are maintained by recycling Ub from its target substrates, when its function is completed, by the rates of Ub degradation and by transcriptional control at four different genetic loci (Park and Ryu, 2014, Shabek et al., 2009). De novo ubiquitin synthesis is achieved in humans thanks to four genes: monomeric Ub-ribosomal fusion genes, UBA52 and RPS27A, that encode one Ub unit fused to a ribosomal protein, and polyubiquitin genes, UBB and UBC, which harbor 3–4 and 9–10 tandem repeats of Ub coding units, respectively (Wiborg et al., 1985, Finley et al., 1989, Baker and Board, 1991). While the Ub-ribosomal fusion genes (also referred to as UBA genes) are constitutively expressed and contribute to fulfill the ubiquitin demand in basal conditions, the polyubiquitin genes have long been known to play a role in stress-response and to be induced when cells are exposed to threats such as heat shock, starvation, DNA damaging agents, proteasome inhibitors, oxidative stress and so on (Finley et al., 1987, Fornace et al., 1989, Finch et al., 1992, Vihervaara et al., 2013).

Although UBB and UBC are transcriptionally upregulated in response to cell stress, both polyubiquitin products also appear to contribute to basal ubiquitin levels, as demonstrated by the phenotypic consequences of targeted homozygous deletion of UBC and UBB in mice (Ryu et al., 2007, Ryu et al., 2008a, Ryu et al., 2008b).

Disruption of polyubiquitin gene UBC leads to mid-gestation embryonic lethality, probably due to an impairment in fetal liver development, which can be only partially rescued by ectopic expression of Ub. Moreover, the decreased Ub content upon UBC disruption is not compensated by upregulation of the other poly- or mono-ubiquitin genes, suggesting that they are functionally not redundant (Ryu et al., 2007, Park et al., 2013). On the other hand, homozygous deletion of UBB in mice caused infertility and adult-onset hypothalamic neurodegeneration with metabolic and sleep abnormalities (Ryu et al., 2008a, Ryu et al., 2008b, Ryu et al., 2010). On the whole, these results prove that a proper expression of polyubiquitin genes is required for normal cell survival and development in mammals. This is in contrast with evidence found in yeast, where the deletion of the stress-inducible polyubiquitin gene UBI4 did not affect cell viability under normal growth conditions, but caused an increased sensitivity towards different stressors, like heat shock and starvation (Finley et al., 1987).

In the present paper, we investigate the contribution of the four Ub genes to the total Ub transcriptome in basal conditions in different cell lines, highlighting the polyubiquitin genes importance for normal cell growth. We also evaluate the responsiveness of Ub genes to different cellular threats (proteasome inhibitors, oxidative stressors, heat-shock, UV) in HeLa cells. Despite the role of specific Ub genes in facing different cell challenges has been previously investigated, studies addressing the whole Ub genes response to different environmental hostile conditions that provoke cellular stress are lacking. Data presented herein provide evidence of the transcriptional program mounted by HeLa cells to increase the ubiquitin supply, needed to counteract both proteotoxic and genotoxic stress. Our results suggest that the ubiquitin-related transcriptional response to stress is cell-type dependent rather than a general response occurring with standard features, in reaction to all types of stress, in all cellular contexts.

Intriguingly, herein we report for the first time the evidence of ubiquitin C mRNA variants, arising from multiple transcription start sites and we characterize and directly compare their expression under basal and stressful conditions.