The images shown in (A,B) are representative of three independent experiments

The images shown in (A,B) are representative of three independent experiments. 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 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 gene by negatively affecting the splicing of its pre-mRNA, providing a straightforward feedback strategy for the homeostatic control of ubiquitin swimming pools. or locus9C12; (3) Ub is present inside the cell primarily partitioned into free and conjugated swimming pools which are not static, but in dynamic equilibrium that changes to meet the changing cellular needs13,14; (4) Ub is one of the most abundant proteins, but remarkably it is not produced in extra, as shown from the upregulation of polyubiquitin coding genes and synthesis of the protein and an improved Ub sparing from proteasomal degradation17,18, a redistribution of ubiquitin from histones to unfolded protein conjugates has been observed19. This competition between different Ub demanding processes displays the limited pool of free Ub. This is also shown by the evidence that, in yeast, Ub depletion may represent the main cause of toxicity induced by translational inhibitors20. Given the involvement of Ub Rabbit Polyclonal to PLCB3 in many different cellular functions (in both normal and stressful conditions), keeping Ub homeostasis is definitely of paramount importance for each and every cell type and requires a highly dynamic but stringent rules. In fact, it has been shown that any alteration in Ub homeostasis, resulting in either an excess or a deficiency of free Ub, causes a ubiquitin stress response21. In particular, elevated Ub levels are intrinsic features of a variety of pathophysiological conditions, that upregulate Ub22C25, but may also derive from exogenous manipulation of cellular Ub levels, Epothilone D leading to ectopic Ub overexpression9,20. In a very recent paper, Han and coworkers26 developed a new system to increase the cellular Ub levels in a more physiological fashion; they used the CRISPR-Cas9 technology to induce upregulation of the endogenous gene under normal conditions. The authors claim that this system may be useful to study the cellular response to an excess of Ub under normal conditions and to highlight if this previous upregulation of may have a protective part towards incoming stress insults. Ubiquitin overexpression has been proved to be protecting in the save from toxicity provoked by inhibitors of translation, which deplete free Ub by reducing its synthesis20. On the other side, alteration of Ub homeostasis in mice, by overexpression of Ub in the neuronal compartment, impaired the synaptic function27. Moreover, 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 manifestation of the endogenous polyubiquitin genes and downregulation in Ub overexpressing cells. Indeed, we found that overexpression of wild-type ubiquitin in different human being cell lines (both normal and tumor derived) resulted in lowered levels of and mRNAs; moreover, the fold-decrease was directly related to the amount of ubiquitin overexpressed, suggesting that a appropriate negative opinions Epothilone D regulatory mechanism, able to sense the Ub levels, could act to keep up Ub within a defined concentration range under unstressed conditions. Another challenging issue is to focus on the and gene manifestation. Results Overexpression of ubiquitin downregulates the endogenous gene manifestation Wild-type ubiquitin (Ubwt) was overexpressed in HeLa cells like a fusion product having a C-terminal Myc-tag, a strategy that reproduces the endogenous manifestation mechanisms28. Previous work has shown that Ub-transfected cells displayed a significantly higher Ub content material (about 4-collapse) compared to cells receiving the bare vector pCMV-Myc or remaining untreated, equally distributed between the free and conjugated swimming pools28. To determine if ubiquitin overexpression experienced effects on its endogenous manifestation, we first examined the mRNA levels of the four Ub coding genes by RTqPCR. No significant changes in the and transcripts were recognized (Fig.?1A). In contrast, ubiquitin overexpression caused a significant decrease (around 50%) in the mRNA levels of the endogenous and genes (Fig.?1A). Transfection of different amounts of Ub create resulted in an increase of total ubiquitin content which was purely correlated to the amount of transgene delivered28 (Fig.?1B). Downregulation of the 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 recognized in pCMV-Myc transfected Epothilone D cells, indicating that this regulatory loop may have a physiological relevance. To investigate whether downregulation upon Ub overexpression was a general buffer mechanism to keep up Ub homeostasis, we.