DECIPHERING THE HUMAN NUCLEOLAR PROTEOME* Yohann Coute ´, 1 * Jennifer A. Burgess, 1 Jean-Jacques Diaz, 2 Christine Chichester, 3 Fre ´de ´rique Lisacek, 3,4,5 Anna Greco, 2 and Jean-Charles Sanchez 1 1 Biomedical Proteomics Research Group, De ´partement de Biologie Structurale et Bioinformatique, Centre Me ´dical Universitaire, 1 Rue Michel Servet, 1211 Geneva 14, Switzerland 2 Centre de Ge ´ne ´tique Mole ´culaire et Cellulaire, CNRS-UMR 5534, Domaine Scientifique de la DOUA, Ba ˆt. Gregor Mendel, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France 3 Geneva Bioinformatics (GeneBio), 25 Av. de Champel, 1206 Geneva, Switzerland 4 Swiss Institute of Bioinformatics, Proteome Informatics Group, 1 Rue Michel Servet, 1211 Geneva 14, Switzerland 5 Ge ´nome & Informatique, Tour Evry 2, 91034 Evry Cedex, France Received 10 March 2005; received (revised) 6 June 2005; accepted 10 June 2005 Published online 4 October 2005 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20067 Nucleoli are plurifunctional nuclear domains involved in the regulation of several major cellular processes such as ribosome biogenesis, the biogenesis of non-ribosomal ribonucleoprotein complexes, cell cycle, and cellular aging. Until recently, the protein content of nucleoli was poorly described. Several proteomic analyses have been undertaken to discover the molecular bases of the biological roles fulfilled by nucleoli. These studies have led to the identification of more than 700 proteins. Extensive bibliographic and bioinformatic analyses allowed the classification of the identified proteins into func- tional groups and suggested potential functions of 150 human proteins previously uncharacterized. The combination of improvements in mass spectrometry technologies, the char- acterization of protein complexes, and data mining will assist in furthering our understanding of the role of nucleoli in different physiological and pathological cell states. # 2005 Wiley Periodicals, Inc., Mass Spec Rev 25:215–234, 2006 Keywords: sub-cellular proteomics; nucleolus; mass spectro- metry; ribosome biogenesis; gene expression regulation I. INTRODUCTION: NUCLEOLI, PLURIFUNCTIONAL DOMAINS OF THE NUCLEUS A. The Cell Nucleus: An Organized and Dynamic Cell Organelle Microscopists first described the nucleus of eukaryotic cells about 200 years ago. It is physically separated from the cytoplasm by a lipid bilayer that acts as a regulator of import and export of molecules through nuclear pores. The nucleus has a high degree of organization. It contains a number of highly dynamic domains, in relation to DNA replication, RNA metabolism and transport (for reviews see Park & De Boni, 1999; Misteli, 2001). The organization of the nucleus depends on the species, cell type, and physiological or pathological states of the cell (Carter et al., 1993; Lewis & Tollervey, 2000; Dundr & Misteli, 2001). During interphase, the nucleus is compartmentalized into domains that support the major biological nuclear activities. Chromosomes are arranged as discrete entities called chromosome territories. Nascent pre-mRNAs localize at the surface of these chromosome territories. The mRNAs are synthesized from active genes within perichromatin fibrils where co-transcriptional splicing also occurs. In the interchromatin space, various molecular species constantly associate and dissociate to give rise to membrane-less nuclear domains whose composition and function have not yet been fully elucidated (Zimber, Nguyen, & Gespach, 2004). At this point in time, approximately 30 different nuclear domains have been characterized including nucleoli, the splicing factor compartments, the Cajal bodies (CBs), and the promyelocytic leukemia (PML) bodies (Dundr & Misteli, 2001). Recently, the high dynamic of the cell nucleus has been demonstrated, notably by use of high resolution fluorescence microscopy and photobleaching techniques (Phair & Misteli, 2000). It was demonstrated that nuclear molecules freely diffuse through the nuclear space and that they are constantly exchanged from their accumulation domain with the nucleoplasm. This was shown for proteins (reviewed in Misteli, 2001) but also for RNAs (Politz et al., 1998, 1999). For example, it was calculated that the mean residence time of fibrillarin within nucleoli was less than 40 sec (Phair & Misteli, 2000). These observations allowed the proposal that nuclear molecules freely diffuse within nucleo- plasm until they find a binding partner with which they interact for a certain time before being released and diffuse again in the nucleoplasm (Misteli, 2001). However, in spite of this continual exchange of molecules between nuclear compartments and the nucleoplasm, nuclear domains are maintained because, at a given time, the number of proteins within a domain is largely superior to the number of proteins that are released from this domain. For instance, it was calculated that approximately 12,000 molecules of fibrillarin leave nucleoli every second out of about 500,000 molecules (Phair & Misteli, 2000). Mass Spectrometry Reviews, 2006, 25, 215–234 # 2005 by Wiley Periodicals, Inc. ———— The supplementary materials referred to in this article can be found at http://www.interscience.wiley.com/jpages/0277-7037/suppmat/. *Correspondence to: Yohann Coute ´, BPRG, De ´partement de Biologie Structurale et Bioinformatique, 1 Rue Michel Servet, 1211 Geneva 14, Switzerland. E-mail: yohann.coute@medecine.unige.ch