Liang Jason, Singh Namit, Carlson Christopher R, Albuquerque Claudio P, Corbett Kevin D, Zhou Huilin
Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, California 92093, USA.
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093, USA.
Genes Dev. 2017 Apr 15;31(8):802-815. doi: 10.1101/gad.296145.117. Epub 2017 May 9.
Post-translational modification by SUMO (small ubiquitin-like modifier) plays important but still poorly understood regulatory roles in eukaryotic cells, including as a signal for ubiquitination by SUMO targeted ubiquitin ligases (STUbLs). Here, we delineate the molecular mechanisms for SUMO-dependent control of ribosomal DNA (rDNA) silencing through the opposing actions of a STUbL (Slx5:Slx8) and a SUMO isopeptidase (Ulp2). We identify a conserved region in the Ulp2 C terminus that mediates its specificity for rDNA-associated proteins and show that this region binds directly to the rDNA-associated protein Csm1. Two crystal structures show that Csm1 interacts with Ulp2 and one of its substrates, the rDNA silencing protein Tof2, through adjacent conserved interfaces in its C-terminal domain. Disrupting Csm1's interaction with either Ulp2 or Tof2 dramatically reduces rDNA silencing and causes a marked drop in Tof2 abundance, suggesting that Ulp2 promotes rDNA silencing by opposing STUbL-mediated degradation of silencing proteins. Tof2 abundance is rescued by deletion of the STUbL or disruption of its SUMO-interacting motifs, confirming that Tof2 is targeted for degradation in a SUMO- and STUbL-dependent manner. Overall, our results demonstrate how the opposing actions of a localized SUMO isopeptidase and a STUbL regulate rDNA silencing by controlling the abundance of a key rDNA silencing protein, Tof2.
小泛素样修饰物（SUMO）介导的翻译后修饰在真核细胞中发挥着重要但仍未被充分理解的调控作用，包括作为SUMO靶向泛素连接酶（STUbL）介导泛素化的信号。在此，我们通过STUbL（Slx5:Slx8）和SUMO异肽酶（Ulp2）的相反作用，阐述了SUMO依赖性控制核糖体DNA（rDNA）沉默的分子机制。我们在Ulp2的C末端鉴定出一个保守区域，该区域介导其对rDNA相关蛋白的特异性，并表明该区域直接与rDNA相关蛋白Csm1结合。两个晶体结构显示，Csm1通过其C末端结构域中相邻的保守界面与Ulp2及其底物之一——rDNA沉默蛋白Tof2相互作用。破坏Csm1与Ulp2或Tof2的相互作用会显著降低rDNA沉默，并导致Tof2丰度显著下降，这表明Ulp2通过对抗STUbL介导的沉默蛋白降解来促进rDNA沉默。通过缺失STUbL或破坏其SUMO相互作用基序可挽救Tof2的丰度，证实Tof2是以SUMO和STUbL依赖性方式被靶向降解的。总体而言，我们的结果证明了局部SUMO异肽酶和STUbL的相反作用如何通过控制关键rDNA沉默蛋白Tof2的丰度来调节rDNA沉默。
