Abstract
Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes.
Supplemental material for this article may be found at http://mcb.asm.org/.
We are very grateful to Christophe Dez, Isabelle Léger-Sylvestre, Jorge Pérez-Fernández, and Olivier Gadal for reagents, protocols, advice, and helpful discussions. We acknowledge Coralie Caron, Valérie Cadamuro, Marie-Françoise O'Donohue, and Pierre-Emmanuel Gleizes for reagents and help with microscopy, cell culture, and pre-rRNA processing in human cells. We also thank all the members of the group of M.C-.F. and Y.H. for advice and helpful discussions. We thank Philippe Pasero (IGH/CNRS, Montpellier, France) for fluorescence-activated cell sorting (FACS) protocols and helpful discussions. We acknowledge Carine Froment and Bernard Monsarrat from the proteomic facility of the Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, Toulouse, France, for mass spectrometry analyses as well as Aurélie Leru and the use of the FACS facility of CBD-IFR109. Anti-Lcp5p antibodies and pUN100-GFP-NOP1 plasmid were generously provided by Thomas Wiederkehr (Basel, Switzerland) and Ed Hurt, respectively. We thank Annie Mougin, Christophe Dez, Jorge Pérez-Fernández, Pierre-Emmanuel Gleizes, and Julien Soudet for critical reading of the manuscript and David Villa for help with figures.
This work was supported by the CNRS, Université Paul Sabatier, and grants from the Agence Nationale de la Recherche and the Ligue Contre le Cancer. M.G. is supported by a Ph.D. fellowship from the Ligue Contre le Cancer. A.K.H. is supported by a Carrier Development Award from the Human Frontier Science Program Organization (HFSPO).