Ubiquitin proteomics identifies RNA polymerase I as a target of the Smc5/6 complex

Ubiquitination controls numerous cellular processes, and its deregulation is associated with 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 elusive. Here, we use label-free quantitative proteomics to analyze the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts the ubiquitination of several proteins involved in ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of Smc5/6. In addition, our analysis suggests a connection between Nse1 and RNA polymerase I (RNA Pol I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote 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 RNA Pol I. [Display omitted] •Proteomics identifies Nse1-sensitive ubiquitin targets beyond canonical Smc5/6 functions•Smc5/6-Nse1 activity promotes ubiquitination of the clamp domain in RNA Pol I•Elongation blocks trigger ubiquitination of Rpa190 at K408/K410•Rpa190 ubiquitination improves rDNA disjunction in smc5/6 mutants Ibars et al. use proteomics to show that the Smc5/6-Nse1 complex affects ubiquitination of proteins beyond its functions in chromosome segregation. In addition, they propose that RNA Pol I elongation problems promote ubiquitination of the Rpa190 subunit to facilitate Smc5/6-dependent separation of the ribosomal DNA locus in mitosis.