Interdependence of Pes1, Bop1, and WDR12 controls nucleolar localization and assembly of the PeBoW complex required for maturation of the 60S ribosomal subunit.
The PeBoW complex is essential for cell proliferation and maturation of the large ribosomal subunit in mammalian cells. Here we examined the role of PeBoW-specific proteins Pes1, Bop1, and WDR12 in complex assembly and stability, nucleolar transport, and pre-ribosome association. Recombinant expression of the three subunits is sufficient for complex formation. The stability of all three subunits strongly increases upon incorporation into the complex. Only overexpression of Bop1 inhibits cell proliferation and rRNA processing, and its negative effects could be rescued by coexpression of WDR12, but not Pes1. Elevated levels of Bop1 induce Bop1/WDR12 and Bop1/Pes1 subcomplexes. Knockdown of Bop1 abolishes the copurification of Pes1 with WDR12, demonstrating Bop1 as the integral component of the complex. Overexpressed Bop1 substitutes for endogenous Bop1 in PeBoW complex assembly, leading to the instability of endogenous Bop1. Finally, indirect immunofluorescence, cell fractionation, and sucrose gradient centrifugation experiments indicate that transport of Bop1 from the cytoplasm to the nucleolus is Pes1 dependent, while Pes1 can migrate to the nucleolus and bind to preribosomal particles independently of Bop1. We conclude that the assembly and integrity of the PeBoW complex are highly sensitive to changes in Bop1 protein levels.
Rohrmoser
Michaela
M
Institute of Clinical Molecular Biology and Tumor Genetics, GSF Research Center, Marchioninistrasse 25, D81377 Munich, Germany.
Hölzel
Michael
M
Grimm
Thomas
T
Malamoussi
Anastassia
A
Harasim
Thomas
T
Orban
Mathias
M
Pfisterer
Iris
I
Gruber-Eber
Anita
A
Kremmer
Elisabeth
E
Eick
Dirk
D
D000818:Animals; D018797:Cell Cycle Proteins; D002458:Cell Fractionation; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism*; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
C488958:Bop1 protein, mouse; D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; C484463:PES1 protein, human; D021122:Protein Subunits; D011506:Proteins; D012322:RNA Precursors; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C517873:WDR12 protein, human