Belli, G. | Search bioRxiv

bioRxiv2021

Ubiquitin proteomics reveals critical targets of the Nse1 RING domain in rDNA and genome stability

Ubiquitination controls numerous cellular processes, and its deregulation is associated to many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and essential functions in genome integrity. However, Nse1-dependent ubiquitin targets remain largely unknown. Here, we use label-free quantitative proteomics to analyse the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts on the ubiquitination of several proteins involved in DNA damage tolerance, ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of the Smc5/6 complex in chromosome disjunction. In addition, our analysis uncovers an unexpected connection between Nse1 and RNA polymerase I (RNAP I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote the ubiquitination of K408 and K410 in the clamp domain of Rpa190, a modification that induces its degradation in response to blocks in transcriptional elongation. We propose that this mechanism contributes to Smc5/6-dependent segregation of the rDNA array, the locus transcribed by RNAP I.

molecular biology↗

bioRxiv2017

Metabolic Constraints And Quantitative Design Principles In Gene Expression During Adaption Of Yeast To Heat Shock

Microorganisms evolved adaptive responses that enable them to survive stressful challenges in ever changing environments by adjusting metabolism through the modulation of gene expression, protein levels and activity, and flow of metabolites. More frequent challenges allow natural selection ampler opportunities to select from a larger number of phenotypes that are compatible with survival. Understanding the causal relationships between physiological and metabolic requirements that are needed for cellular stress adaptation and gene expression changes that are used by organisms to achieve those requirements may have a significant impact in our ability to interpret and/or guide evolution.\n\nHere, we study those causal relationships during heat shock adaptation in the yeast Saccharomyces cerevisiae. We do so by combining dozens of independent experiments measuring whole genome gene expression changes during stress response with a nonlinear simplified kinetic model of central metabolism.\n\nThis combination is used to create a quantitative, multidimensional, genotype-to-phenotype mapping of the metabolic and physiological requirements that enable cell survival to the feasible changes in gene expression that modulate metabolism to achieve those requirements. Our results clearly show that the feasible changes in gene expression that enable survival to heat shock are specific for this stress. In addition, they suggest that genetic programs for adaptive responses to desiccation/rehydration and to pH shifts might be selected by physiological requirements that are qualitatively similar, but quantitatively different to those for heat shock adaptation. In contrast, adaptive responses to other types of stress do not appear to be constrained by the same qualitative physiological requirements. Our model also explains at the mechanistic level how evolution might find different sets of changes in gene expression that lead to metabolic adaptations that are equivalent in meeting physiological requirements for survival. Finally, our results also suggest that physiological requirements for heat shock adaptation might be similar between unicellular ascomycetes that live in similar environments. Our analysis is likely to be scalable to other adaptive response and might inform efforts in developing biotechnological applications to manipulate cells for medical, biotechnological, or synthetic biology purposes.

systems biology↗