Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis.
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.
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: skawashima@mol.f.u-tokyo.ac.jp.
Shigehiro A
SA
Kawashima
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA.
Zhen
Z
Chen
The University of Tokyo, Graduate School of Pharmaceutical Sciences, Tokyo 1130033, Japan.
Yuki
Y
Aoi
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA.
Anupam
A
Patgiri
The University of Tokyo, Graduate School of Pharmaceutical Sciences, Tokyo 1130033, Japan.
Yuki
Y
Kobayashi
Laboratory of Yeast Genetics and Cell Biology, The Rockefeller University, New York, NY 10065, USA; The Francis Crick Institute, London NW1 2BE, UK.
Paul
P
Nurse
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address: kapoor@rockefeller.edu.
Tarun M
TM
Kapoor
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D007211:Indoles / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D054683:Ribosome Subunits, Large, Eukaryotic / Q000187:drug effects* / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000187:drug effects / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D014227:Triazines / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D007211:Indoles; D029702:Schizosaccharomyces pombe Proteins; D014227:Triazines; D000251:Adenosine Triphosphatases
AAA(+) protein; Midasin; Schizosaccharomyces pombe; chemical genetics; chemical inhibitors; chemical screen; fission yeast; ribosome biogenesis
Nat Chem Biol. 2012 Feb 12;8(3):235-7
22327403
BMC Genomics. 2002 Jul 8;3:18
12102729
Yeast. 1998 Jul;14 (10 ):943-51
9717240
Chem Biol. 2013 Feb 21;20(2):262-71
23438755
J Biol Chem. 2004 Dec 31;279(53):55411-8
15528184
J Biol Chem. 2014 Feb 14;289(7):3913-22
24371142
EMBO J. 2002 Oct 15;21(20):5539-47
12374754
Nat Rev Mol Cell Biol. 2005 Jul;6(7):519-29
16072036
J Cell Sci. 2013 Feb 1;126(Pt 3):705-13
23525020
Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):4834-9
21383145
J Cell Sci. 2016 Mar 15;129(6):1250-9
26869222
Trends Cell Biol. 1998 Feb;8(2):65-71
9695811
Cell Cycle. 2014;13(8):1327-34
24621506
Mol Cell. 2010 Jun 11;38(5):712-21
20542003
BMC Genomics. 2003 Jul 10;4(1):27
12854975
EMBO J. 1996 Dec 2;15(23):6617-28
8978688
Nat Struct Mol Biol. 2015 Jun;22(6):492-8
25938660
Elife. 2014 Sep 18;3:e03574
25233066
Annu Rev Biophys Biomol Struct. 2006;35:93-114
16689629
Chem Biol. 2012 Jul 27;19(7):893-901
22840777
Trends Biochem Sci. 1999 Nov;24(11):437-40
10542411
Biochim Biophys Acta. 2012 Jan;1823(1):92-100
21763358
J Cell Biol. 1999 Feb 8;144(3):389-401
9971735
Methods Enzymol. 1991;194:795-823
2005825
Biochemistry. 2004 Sep 7;43(35):11266-74
15366936
Nature. 2014 Jan 2;505(7481):112-6
24240281
Cancer Cell. 2015 Nov 9;28(5):653-65
26555175
Mol Cell. 2004 Jul 23;15(2):295-301
15260980
Trends Cell Biol. 2003 May;13(5):255-63
12742169
J Cell Sci. 2013 Nov 1;126(Pt 21):4815-21
24172536
Nature. 2012 Mar 18;484(7392):125-9
22425997
Cell. 2009 Sep 4;138(5):911-22
19737519
EMBO J. 1986 Nov;5(11):2973-9
16453724