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

Shigehiro A. Kawashima; Zhen Chen; Yuki Aoi; Anupam Patgiri; Yuki Kobayashi; Paul Nurse; Tarun M. Kapoor

doi:10.1016/j.cell.2016.08.070

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

Shigehiro A. Kawashima^*, Zhen Chen, Yuki Aoi, Anupam Patgiri, Yuki Kobayashi, Paul Nurse, Tarun M. Kapoor

^*Corresponding author for this work

Biochemistry and Molecular Genetics

Research output: Contribution to journal › Article › peer-review

53 Scopus citations

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.

Original language	English (US)
Pages (from-to)	512-524.e14
Journal	Cell
Volume	167
Issue number	2
DOIs	https://doi.org/10.1016/j.cell.2016.08.070
State	Published - Oct 6 2016

Funding

We thank Hiro-oki Iwakawa and Yukihide Tomari for technical advice and help with northern blot experiments, Kuniko Saiki for help with northern blot experiments, Yoshinori Watanabe and Jun-ichi Nakayama for providing strains and plasmids, Akihisa Matsuyama and Minoru Yoshida for providing plasmids, Fraser Glickman for the use of the Rockefeller University High Throughput Screening Resource Center, Milica Tesic Mark from Rockefeller Proteomics Resource Center, Olivier Elemento for help with the bioinformatics analysis, Jonathan Steinman for help with analog synthesis, and Sebastian Klinge and Lisa Hang for valuable comments. This work was supported by JSPS KAKENHI Grant Number JP26711001 (to S.A.K.), Helen Hay Whitney Fellowship (to A.P.), the Breast Cancer Research Foundation (to P.N.), the Wellcome Trust (to P.N.), and the NIH/NIGMS GM98579 (to T.M.K.).

Keywords

AAA protein
chemical genetics
chemical inhibitors
chemical screen
fission yeast
Midasin
ribosome biogenesis
Schizosaccharomyces pombe

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

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10.1016/j.cell.2016.08.070

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title = "Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis",

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{\textquoteright} 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.",

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author = "Kawashima, {Shigehiro A.} and Zhen Chen and Yuki Aoi and Anupam Patgiri and Yuki Kobayashi and Paul Nurse and Kapoor, {Tarun M.}",

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AU - Nurse, Paul

AU - Kapoor, Tarun M.

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N2 - 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.

AB - 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.

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

Abstract

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Keywords

ASJC Scopus subject areas

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