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

Summary 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.
Graphical Abstract
Highlights • Ribozinoindoles are potent chemical inhibitors of eukaryotic ribosome assembly • Activity of four of Mdn1’s six ATPase sites is likely needed for cell growth • Ribozinoindoles inhibit recombinant full-length Mdn1’s ATPase activity in vitro • Assembly of Nsa1 particles, precursors of the 60S subunit, depends on Mdn1
Selective potent inhibitors of eukaryotic ribosome biogenesis are found through a chemical synthetic lethal screen.