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Nucleolin Structure and Functions 763 Journal of Cell Science 112, 761-772 (1999) 761 Printed in Great Britain © The Company of Biologists Limited 1999 JCS5011 COMMENTARY Structure and functions of nucleolin Hervé Ginisty*, Hélène Sicard*, Benoit Roger and Philippe Bouvet‡ Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse Cedex, France *These authors contributed equally to this work ‡Author for correspondence at present address: CNSR-IPBS, UPR 90062, 205 route de Narbonne, 31077 Toulouse, France (E-mail: [email protected]). Published on WWW 25 February 1999 SUMMARY Nucleolin is an abundant protein of the nucleolus. cytoplasmic transport. Studies of nucleolin over the last 25 Nucleolar proteins structurally related to nucleolin are years have revealed a fascinating role for nucleolin in found in organisms ranging from yeast to plants and ribosome biogenesis. The involvement of nucleolin at mammals. The association of several structural domains in multiple steps of this biosynthetic pathway suggests that it nucleolin allows the interaction of nucleolin with different could play a key role in this highly integrated process. proteins and RNA sequences. Nucleolin has been implicated in chromatin structure, rDNA transcription, Key words: Nucleolin, Ribosome biogenesis, Nucleolus, Ribosomal rRNA maturation, ribosome assembly and nucleo- RNA, RNA-binding protein INTRODUCTION dimensional gel (Prestayko et al., 1974). The same protein was then described and purified from Chinese Hamster Ovary cells Since the discovery of the nucleolus, many works have focused (Bugler et al., 1982) and several other eukaryotic cells. The on understanding the function of this dynamic nuclear name nucleolin is now widely used for this nucleolar protein, structure. Ribosome biogenesis is the major function known to which can represent as much as 10% of total nucleolar protein be associated with the nucleolus (Hadjiolov, 1985), although in CHO (Bugler et al., 1982). This protein is also often recent work suggest that this nuclear compartment might be described as a 100-110 kDa protein; however, cloning of the involved in other cellular processes (Pederson, 1998). cDNA of hamster nucleolin revealed that it contained 713 The nucleolus is the site of ribosomal RNA (rRNA) amino acids, giving rise to a predicted molecular mass of 77 transcription, rRNA modification, maturation and assembly kDa (Lapeyre et al., 1987, 1985). This discrepancy was latter with ribosomal proteins to form the pre-ribosomal particles that attributed to the amino acid composition of the N-terminal are then exported to the cytoplasm to form the mature domain of nucleolin. Homologous proteins were then identified ribosomes. Ribosome biogenesis is probably one of the most in man (Srivastava et al., 1989), rat (Bourbon et al., 1988), complex pathways of ribonucleoparticle synthesis, involving a mouse (Bourbon et al., 1988), chicken (Maridor and Nigg, complex rRNA maturation and an ordered assembly of about 1990) and Xenopus laevis (Caizergues-Ferrer et al., 1989; 80 ribosomal proteins in eukaryotes. In addition to the Rankin et al., 1993). Nucleolin is highly phosphorylated component found in the mature ribosomes, the nucleolus (Olson et al., 1975; Rao et al., 1982) and methylated (Lischwe contains many other RNAs and proteins, most of which et al., 1982), and could also be ADP-ribosylated (Leitinger and transiently associate with pre-ribosomal particles and appear to Wesierska-Gadek, 1993). be involved in various aspects of ribosome biogenesis The human nucleolin gene is present at one copy per haploid (transcription, maturation, modification and ribosome genome. The human gene consists of 14 exons and 13 introns assembly). In this review we focus on one of the major and on chromosome 2q12-qter (Srivastava et al., 1990). The four most studied nucleolar proteins, nucleolin, whose multiple acidic stretches of the N-terminal domain lie within exons 2 to functions in ribosome biogenesis are beginning to be 4 and the nuclear localization signal (NLS) is encoded in exon characterized. 5. Interestingly, each of the four RNA-binding domains is encoded by two independent and consecutive exons. For the Properties of the different domains of nucleolin and four RNA-binding domains, the intron position is similar and ‘nucleolin-like proteins’ interrupts the conserved RNP1 sequence of each RNA-binding Nucleolin was first described by Orrick et al. (1973) and was domain, suggesting that these RNA-binding domains are initially called C23 because of its mobility on a two- duplications of the same ancestor RBD domain. This genomic 762 H. Ginisty and others organization is extremely well conserved in hamster, mouse including casein kinase II (CK2) (Caizergues-Ferrer et al., and rat (Bourbon and Amalric, 1990; Bourbon et al., 1988). 1987), p34cdc2 (Belenguer et al., 1989; Peter et al., 1990) and Another intriguing feature of nucleolin gene organization (in protein kinase C-ζ (Zhou et al., 1997). CK2 co-purifies with tetrapod and vertebrates) is that two snoARNs (U20 and U23 nucleolin (Caizergues-Ferrer et al., 1987), and recent work snoRNA) are encoded in introns 11 and 12, respectively indicates that α or α′ subunits of CK2 interact directly with (Nicoloso et al., 1994; L. H. Qu and J. P. Bachellerie, personal nucleolin (Li et al., 1996). CK2 phosphorylates nucleolin in communication). U20 and U23 belong to the two large families vitro and in vivo at serine residues found predominantly in two of snoRNAs, the box C/D antisense snoRNAs and the H/ACA highly acidic regions (Caizergues-Ferrer et al., 1987). p34cdc2 snoRNAs, respectively, which guide the two major types of phosphorylation occurs on threonine residues within the basic rRNA nucleotide modifications, namely ribose methylation TPXKK repeat (Belenguer et al., 1989; Peter et al., 1990). All and pseudouridine formation (Bachellerie and Cavaillé, 1998; potential sites for p34cdc2 kinase are not used with the same Smith and Steitz, 1997). In vertebrates, most modification- efficiency in vivo as in vitro (Belenguer et al., 1989). guiding snoRNAs are intron-encoded and processed from pre- Phosphorylation of nucleolin by CK2 and p34cdc2 is highly mRNA introns, and their host genes are usually directly regulated during the cell cycle (Belenguer et al., 1989; Peter et involved in ribosome biogenesis (Bachellerie and Cavaillé, al., 1990). Extensive phosphorylation by CK2 occurs in 1998; Bachellerie et al., 1995). interphase and by p34cdc2 in mitosis, and this regulated Analysis of the amino acid sequence of nucleolin reveals the phosphorylation of nucleolin probably regulates nucleolin presence of three different structural domains (Fig. 1). The N- function during the cell cycle. terminal domain is made up of highly acidic regions In plants and yeast, phosphorylation sites in the N-terminal interspersed with basic sequences and contains multiple domain have been diversely conserved. Nucleolin-like proteins phosphorylation sites. The central domain contains four RNA- from plants (Bogre et al., 1996; de Carcer et al., 1997) and binding domains called RBD or RRM. The C-terminal domain yeast (Gulli et al., 1997) exhibit consensus CK2 called GAR or RGG domain is rich in glycine, arginine and phosphorylation sites, or are highly phosphorylated by this phenylalanine residues. This domain contains high levels of kinase. Plant nucleolin-like proteins are also phosphorylated by NG,NG-dimethylarginines (Lischwe et al., 1982). p34cdc2. Although S. pombe gar2 is most likely phosphorylated Several nucleolar proteins of different eukaryotic species by p34cdc2 in mitosis on a single serine residue (Gulli et al., exhibit a similar tripartite structural organization (Table 1 and 1997), a gar2 protein with this serine changed into a non- see legend of Fig. 1 for details). In this review, we will call phosphorylated alanine is able to complement perfectly a gar2- these proteins ‘nucleolin-like proteins’. This does not imply that they are all orthologs of nucleolin in these different species; however, based on what is known of nucleolin’s Central domain properties, we can propose that all these proteins are multifunctional nucleolar proteins involved in different aspects N- domain C- domain of ribosome biogenesis. A Hamster nucleolin N-terminal domain The length of this domain is very variable among the different nucleolin-like proteins (Fig. 1). The presence of highly acidic regions separated from each other by basic sequences is one B NucMs1 (Alfalfa) feature of this domain. Yeast gar2 and Nsr1p N-terminal domains have long stretches of serine residues in the acidic regions. Acidic domains have been proposed to bind histone H1 (Erard et al., 1988), and could be responsible for a displacement of H1 from its interaction with linker DNA. This C gar2 (S. pombe) interaction with histone H1 would induce chromatin decondensation (Erard et al., 1988). These acidic stretches also N- domain determine the Ag-NOR stainability of nucleolin (Roussel et al., 1992). The presence of the basic and repeated octapeptide Acidic stretches motifs (XTPXKKXX, X being a non-polar residue) bears strong similarity to an analogous sequence of histone H1 NLS (Erard et al., 1990). These motifs could be responsible for the capacity of nucleolin to modulate DNA condensation in RNA-binding domain (RBD) chromatin (Erard et al., 1990; Olson and Thompson, 1983). In GAR domain addition to its interaction with chromatin and histone H1, the N-terminal domain of nucleolin has been involved in many protein-protein interaction, such as with the U3 snoRNP Fig. 1. Schematic representation of the organization of three nucleolin and ‘nucleolin-like proteins’. RBDs are defined from the β1 and β4 (Ginisty et al., 1998) and some ribosomal proteins (Bouvet et strand (Kenan et al., 1991). (A) Organization of hamster nucleolin: al., 1998; Sicard et al., 1998). mouse, rat chicken, human and Xenopus laevis nucleolin have the The N-terminal domain of nucleolin is highly same organization. (B) Alfalfa and pea ‘nucleolin-like protein’ have phosphorylated (Bourbon et al., 1983; Mamrack et al., 1979; the same organization. (C) Organization of gar2.
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