Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis

  • Cell. 2016 Oct 6;167(2):512-524.e14. doi: 10.1016/j.cell.2016.08.070.
Shigehiro A Kawashima  1 ,  Zhen Chen  2 ,  Yuki Aoi  3 ,  Anupam Patgiri  2 ,  Yuki Kobayashi  3 ,  Paul Nurse  4 ,  Tarun M Kapoor  5
Affiliations
  • 1. 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. Electronic address: [email protected].
  • 2. Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA.
  • 3. The University of Tokyo, Graduate School of Pharmaceutical Sciences, Tokyo 1130033, Japan.
  • 4. Laboratory of Yeast Genetics and Cell Biology, The Rockefeller University, New York, NY 10065, USA; The Francis Crick Institute, London NW1 2BE, UK.
  • 5. Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address: [email protected].
PMID: 27667686 DOI: 10.1016/j.cell.2016.08.070
Abstract

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.

Keywords
AAA(+) protein; Midasin; Schizosaccharomyces pombe; chemical genetics; chemical inhibitors; chemical screen; fission yeast; ribosome biogenesis.
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