Kawashima Shigehiro A, Chen Zhen, Aoi Yuki, Patgiri Anupam, Kobayashi Yuki, Nurse Paul, Kapoor Tarun M
The University of Tokyo, Graduate School of Pharmaceutical Sciences, Tokyo 1130033, Japan; Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA.
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA.
Cell. 2016 Oct 6;167(2):512-524.e14. doi: 10.1016/j.cell.2016.08.070. Epub 2016 Sep 22.
All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.
所有细胞蛋白质均由核糖体合成，真核生物中核糖体的生物合成是一个在数分钟内完成的复杂多步骤过程。有几种核糖体功能的化学抑制剂可供使用，并用作工具或药物。相比之下，我们缺乏经过有效验证的化学探针来分析真核生物核糖体组装的动态过程。在这里，我们结合化学和遗传方法来发现核糖嗪吲哚（或Rbins），这是一种基于三嗪吲哚的强效且可逆的真核生物核糖体生物合成抑制剂。对裂殖酵母中Rbin敏感性和抗性赋予突变的分析，以及对重组蛋白的生化分析，提供了证据表明Rbins的生理靶点是Midasin，一种必需的约540 kDa的AAA +（与多种细胞活动相关的ATP酶）蛋白。使用Rbins急性抑制或激活Midasin功能，在对抑制剂敏感或抗性细胞的平行实验中，我们揭示了Midasin在组装Nsa1颗粒（60S亚基的核仁前体）中的作用。总之，我们的研究结果表明Rbins是用于真核生物核糖体组装的强大探针。
