The nucleolus

Ulrich Scheer and Dieter Weisenberger

University of Würzburg, Würzburg, Germany

The transcriptionally active rRNA genes have the remarkable ability to organize and integrate the biochemical pathway of ribosome production into a structural framework, the nucleolus. The past year has seen numerous advances in our understanding of the relationships between nucleolar substructures, the site of ribosomal RNA (rRNA) gene transcription and the pathway of ribosome maturation. Progress has also been made both in the molecular identification of nucleolar constituents and in our understanding of the interactions between these components and their assembly into higher order structu res.

Current Opinion in Cell Biology 1994, 6:3 54-359

Introduction Molecular architecture of the nucleolus

Nucleoli are readily visualized by light and electron Most nucleoli displaya concentric arrangement of three microscopy as solitary or multiple non-membranous structural components (Fig. 1). The central element is nuclear structures that occupy a substantial portion a pale-staining region, the fibrillar center, which con­ of the total nuclear interior (Fig. 1). Within nucleoli tains RNA polymerase I, DNA topoisomerase I, and takes place the production of pre-ribosomal compo­ the transcription factor UBF [13,15,16], Fibrillar cen­ nents, including the transcription of the rRNA genes, ters are surrounded, either wholly or in part, by a the processing of the primary transcripts into mature compact layer termed the dense fibrillar component. 18S, 5.8S and 28S rRNAs, the addition of proteins to A major constituent of this region is fibrillarin, a pro­ the nascent pre-ribosomes, and the incorporation of tein associated with several snoRNAs, and involved the 5S RNA, which is synthesized outside of the nu­ in the post-transcriptional assembly of pre-ribosomes cleolus [1). In addition to the rRNA gene clusters and [17--]. The outermost layer has a grainy appearance flanking sequences, the nucleolus harbors a large num­ and is hence termed the granular component. Pulse­ ber of protein and RNA components which are not chase labeling and in situ hybridization experiments part of mature cytoplasmic ribosomes, but rather are have clearly demonstrated that pre-ribosome biogene­ involved in the transcriptional [2,3] and post-transcrip­ sis is a vectorial process. Nascent pre-ribosomes move tional [4,5] regulation of ribosome synthesis. Among from the fibrillar area to the granular component of the these are small nucleolar RNAs (snoRNAs; [6,7]), non-ri­ nucleolus, and are then released into the nucleoplasm bosomal proteins that may be components of nucleolar as nearly mature ribosomal subunits (e.g. [1,11,18]). lt structural elements, proteins that are transiently associ­ is still not clear, however, whether the various events ated with pre-ribosomes, and nucleocytoplasmic shut­ in ribosome maturation can be strictly assigned to par­ tle proteins. ticular nucleolar structures. The nucleolus may contain either a number of spatially fixed 'stations' where spe­ Nucleoli undergo extensive structural changes du ring cific events of pre-rRNA processing and/or assembly the cell cycle of higher eukaryotes; they disappear at take place as the pre-ribosomes pass by, or alterna­ prophase and reappear at telophase. This dynamic be­ tively, each transcript may carry its own processing havior is useful in the analysis of how the various nu­ machinery that functions independently of its specific cleolar components interact with one another to build position within the nucleolus. the intricate structure of the nucleolus. Recent reviews have summarized the organization of the various nu­ The intranucleolar site of ribosomal DNA (rDNA) cleolar structures and their possible functional links to transcription is still under serious debate (for possible ribosome biosynthesis (e.g. [1 ,4,8-14]). In this review models, see [10]). By using a variety of cytochemi­ we discuss some re cent progress in our understanding cal and immunocytochemical approaches, rDNA has of nucleolar architecture, its assembly and its relation­ been localized either to the fibrillar centers exclusively ship to nucleolar function as reported in the preceding [19--,20,211, to the fibrillar center and the proximal re­ year. gions of the surrounding den se fibrillar component

Abbreviations NLS-nuclear localization signal; NOR-nucleolus organizer region; PNB-pre-nucleolar body; rDNA-ribosomal DNA; rRNA-ribosomal RNA; snoRNA-small nucleolar RNA;

354 © Current Biology Ud ISSN 0955-0674 The nudeolus Scheer and Weisenberger 355

'Christmas trees' and nucleolar structure

Nascent transeripts are anchored by dosely spaced 10-14 nm partides containing RNA polymerase I to the chromatin axes of rRNA genes, and their free 5'-ends carry an approximately 25 nm thick granule, the 'terminal ball'. These morphological features are readily seen in Miller-type spread preparations, even when the transcribing rRNA genes (in the form of the characteristic 'Christmas trees') are dosely juxtaposed (Fig. 2). The terminal balls are the ultrastructural coun­ terparts of pre-rRNA processing complexes, consisting of U3 snoRNA and a number of proteins which co-as­ semble dose to the 5'-end of the nascent pre-rRNA [28--). These complexes mediate the U3-dependent first deavage within the 5'-external transcribed spacer in species as distant as mouse and Xenopus [29--). In Miller spreads, the rDNA of transcribed nucleolar genes is in an extended B-form, in which a single mammalian rRNA transcription unit, with a length of ",4 JlI11, could easily span the entire nucleolus in situ (8). Thus, the rRNA genes must be packed extremely tightly within the nudeolus of a living cel!. It is re­ markable that transcribing rRNA genes have not yet been directly visualized in ultrathin sections of cells fixed in situ. Although we do not know the specific conformation of the nascent transcripts with their ter­ minal processing complexes in vivo, it should be fea­ sible in the future to identify active rRNA genes in ul­ trathin sections of nudeoli, using refined methods such as electron microscope tomography.

Nucleolar targeting Fig. 1. Electron micrograph of a human bladder carcinoma cell. Each of the multiple nucleoli is subdivided into three components described in the text. Embedded in the main body of the nucleolus Once transported into the nucleus, how are nucle­ (the granular component) are rounded structures of low contrast, olar proteins recruited to the nucleolus? Evidence is the fibrillar centers. These are surrounded by a compact layer of accumulating that recruitment is not due to a com­ the dense fibrillar component. Bar indicates 111m. mon nucleolar targeting signal, but rather to multiple functional interactions with other macromolecules al­ [22-24), or predominantly to the dense fibrillar com­ ready present in the nucleolus. A quantitatively ma­ ponent (25). To settle the present controversy about jor nucleolar protein, nucleolin, is known to bind to where the rDNA is located, improved DNA localization pre-rRNA and is thought to be involved in its process­ methods are required which combine high sensitivity ing, or in the early stages of ribosome assembly (4). As with optimal preservation of biological structures (see shown by deletional analyses, nucleolin is, like most [19--]). karyophilic proteins, transported into the nucleus by a specific nudear localization signal (NLS). Nucleolar Using an alternative approach, results from the incor­ targeting, however, requires two additional domains poration of UTP analogs followed by immunogold which are capable of interacting with RNA [30--32-). electron microscopy have suggested that rRNA gene The combination of an RNA recognition motif (RRM), transcription takes place at the boundary between the glycine/arginine rich (GAR) domain, and the NLS the fibrillar center and the dense fibrillar component were sufficient to direct a hybrid protein to the nu­ [26-,27-). According to the model proposed by Hozak cleolus ([32-); see, however, [30-,31-]). The nucleolar et a/. [27-), the rDNA transcription units are attached localization of the yeast NSR1 protein, which is related through active RNA polymerases to the surface of the to mammalian nucleolin, also appears to be mediated fibrillar centers, which serve both as the central skeletal by NSR1 protein domains interacting with other nucle­ element of the nudeolus and as a storage site for dis­ olar components [33-) . engaged components of the transcription machinery, such as RNA polymerase land DNA topoisomerase I. Molecular interactions between the nucleolar targeting This view emphasizes the importance of the boundary domain of Rex, a regulatory protein of the human retro­ region between the fibrillar center and the dense fib­ virus HTLV-I, and nucleolar constitutents have been rillar component, and importantly, is reconcilable with studied by affinity chromatography [34-). The nucleolar most previous localization studies. protein B23, a putative ribosome-assembly factor, is the 356 Nudeus and gene expression

Fig. 2. Ribosomal RNA genes in full transcriptional activity, as seen in a spread preparation of an oocyte of the salamander Pleurodeles waltl. The high packing density of the genes probably reflects their in vive situation. Each transcription unit is characterized by closely spaced transcription complexes and the terminal balls of the nascent ribonucleoprotein fibrils. Bar indicates 1 J.lm.

major binding host for Rex. Interestingly, protein B23 acid interactions are involved in establishing and main­ (and its amphibian counterpart N038) belongs to the taining the intricate nucleolar architecture. This con­ group of nucleolar proteins that shuttle between the cept has received further support from the observa­ nucleolus and the cytoplasm. Hence, it is conceivable tion that solubilized components of amoebae nucleoli that the Rex protein can 'piggyback' · into the nucleo­ can reassociate in vitro into nucleolus-like structures 0 00 lus simply by interacting with B23 [34 1. In support with granular and dense fibrillar components [40 ). of this idea, other shuttling nucleolar proteins such But what is the primary determinant of nucleolar or­ as rat liver Nopp140 [351, mammalian nucleolin and ganization? It is now weil established that a single its yeast counterpart, NSR1 [36 0 1, recognize the NLS rRNA gene is sufficient to organize a nucleolus [411, and of karyophilic proteins and may thus provide a shut­ that the structural integrity of a nucleolus is dependent tle service for nucleolar accumulation (as weil as a upon the transcriptional activity of the rRNA genes [8). mechanism for the export of ribosomal subunits). Thus, a single rDNA transcription unit has the potential Taken together, these and earlier nucleolar targeting to organize and maintain nucleolar structure. In prin­ experiments with the protein N038/B23 ([371; see cipal, rDNA-associated proteins, such as transcription also [38]) and the rDNA transcription factor UBF [391 factors or transcribing polymerases, nascent pre-rRNAs, have indicated that different nucleolar proteins are or proteins and snoRNAs that are bound to them could sequestered in the nucleolus by multiple pathways, be involved in this activity. It has been proposed that which depend on the particular binding host within the 5'-ends of the nascent pre-rRNA transcripts may the nucleolus. This mayaiso be true for the nucleo­ be involved in the formation and maintenance of the lar accumulation of RNAs or ribonucleoproteins such dense fibrillar component, which could, in turn, pro­ as 5S rRNA and various snoRNPs. vide the necessary binding sites for elements of the granular component [8,13,42). However, recent results obtained from a mutant yeast strain which synthesizes Maintenance of nudeolar structure rRNA exclusively by RNA polymerase 11 have indicated The nucleolar targeting experiments have indicated that RNA polymerase I itself, rather than the growing that a cascade of protein-protein and protein-nucleic transcripts, may playa role as a structural element for The nucleolus Scheer and Weisenberger 357 nucleolar architecture [43--). These polymerase I dele­ tion yeast mutants contain fibrillarin-positive dense bodies (termed mini-nucleolar bodies) which resem­ ble the dense fibrillar component fragments induced in manm1alian cells after inhibition of rDNA transcrip­ tion [8,44). Oakes et al. [43--) have thus come to the provocative conclusion that an intact nucleolar stmc­ ture is not absolutely required for ribosome biosyn­ thesis, but may simply enhance the efficiency of this process. Perhaps the yeast polymerase I mutant system may provide clues as to why eukaryotes have devel­ oped a nucleolus despite the fact that their prokary­ otic ancestors were perfectly capable of synthesizing ribosomes in the absence of such a structure.

The nucleolus organizer and pre-nucleolar bodies

During mitosis, rRNA synthesis is downregulated; yet proteins that play a central role in rDNA transcrip­ tion, such as RNA polymerase I, DNA topoisomerase I, and UBF remain associated with the chromosomal nu­ cleolus organizer regions (NORs), which also harbor the rRNA genes (13). The mechanisms that modulate the activity of rRNA genes du ring mitosis may provide an interesting case by which to analyze the control of gene activity and stmcture. Postmitotic reformation of nucleoli is dependent on the interaction of two sep­ arate entities, the NOR and the pre-nucleolar bodies (PNBs). PNBs appear at telophase, and are initially scattered throughout the reforming daughter nuclei as they progress into the GI phase (Fig. 3). The PNBs con­ tain a number of proteins characteristic for the den se fibrillar component of interphase nuclei [451, as weil as U3 snoRNA [421. PNB-like stmctures can also form in vitra during the assembly of nuclei in Xenapus egg ex­ tract independent of the presence of rDNA sequences (45). Interestingly, the 'synthetic' PNBs share several Fig. 3. Nucleoli reform after mitosis by the coalescence of pre­ formed entities, the PNBs. (a) A phase contrast image (note the components with coiled bodies such as spliceosomal residual cytoplasmic bridge between the daughter cells, arrow) small nuclear (sn) RNAs and the protein coilin [46-1, and (b) Hoechst staining are shown. (c) PNBs are visualized by indicating that there is a possible relationship between immunofluorescence microscopy of Xenopus A6 cells with mAb these nuclear stmctures. Coiled bodies, which, like the No-114, which recognizes a protein of the dense fibrillar compo­ PNBs, contain fibrillarin and disassemble during mi­ nent of interphase nucleoli (for references see [45]). Bar indicates 10 j1ITl. tosis, often reveal a close physical association with nucleoli [47,48). When the rRNA genes resume their tural framework, the nucleolus. We presently believe transcriptional activity, PNBs fuse around the NOR of that the transcribing rRNA genes serve as a nucleation the developing nucleolus [13). In HeLa cells, however, site where the primary binding and assembly of nucle­ the postmitotic reformation of PNBs and coiled bodies olar components occurs. Do these, in turn, provide the follows different kinetics, and there is currently no evi­ next level of bin ding sites for other nucleolar proteins dence that they are assembled from a common precur­ or RNAs necessary for higher order organization? Are sor stmcture [49). The identification of the components the active RNA polymerase I molecules, the r.ascent involved in this selective targeting process of the PNBs, transcripts or the rDNA-containing chromatin the de­ and their specific mode of interaction, will be impor­ terminants of nucleolar higher order stmcture? Answers tant for understanding the dynamic rearrangements of should co me in the near future from the use of genetic nucleoli during mitosis. approaches in yeasts [43--1 and from refinements in cell-free systems presently available [40--,45,46-). Conclusion Wh at is a nucleolus? Is it just a cloud of transcrip­ ti on products with associated proteins and factors that The transcriptionally active rRNA genes have the re­ gather around the transcribing rRNA genes? Or are markable ability to organize and integrate the bio­ stmctural elements involved in nucleolar function, such chemical pathway of ribosome production into a stmc- as the skeletal network extending throughout the am- 358 Nucleus and gene expression

plified nucleoli of Xenopus [50)? Future progress in un­ 17. Tollervey D, Lehtonen H, ]ansen R, Kern H, Hurt EC: derstanding nucleolar function and ribosome biosyn­ 00 Temperature-Sensitive Mutations Demonstrate Roles for thesis will depend on finding answers to these ques­ Yeast Fibrillarin in Pre-rRNA Processing, Pre-rRNA Methy­ lation, and Ribosome Assembly. Gell 1993, 72:443-457. tions. Yeast fibrillarin (NOPl) is structurally and functionally homologous to the corresponding vertebrate nucleolar protein. ßy using genetic approaches it is shown that fibrillarin is a multifunctional protein in­ Acknowledgements volved in a number of post-transcriptional steps of ribosome syn­ thesis. These are pre-rRNA processing, pre-rRNA modification and We thank Hilde Merkert for providing Fig. 1, Drs D Lourim and R assembly of the rRNA with ribosomal proteins. It is Iikely that verte­ ßenavente for critical comments on this manuscript and our col­ brate fibrillarin carries out the same functions. leagues for communicating results before publication. The authors' 18. Puvion-Dutilleul F, Mazan S, Nicoloso M, Pichard E, Bachel­ research is supported by a gr.mt from the Deutsche Forschungs­ lerie ]-P, Puvion E: Aiterations oe Nucieolar Ultrastructure gemeinschaft (Sche 157/8-2). and Ribosome Biogenesis by Actinomycin D. Implications for U3 snRNP Function. Eur J Gell Bio/ 1992, 58:149-162. 19. Thiry M, Ploton D, Me'nager M, Goessens G: Ultrastructural References and recommended reading 00 Distribution of DNA within the Nucieolus oC Various Animal Cell Lines or Tissues Revealed by Terminal Deoxynucieotidyl Papers of particular interest, published within the annual period of TransCerase. Gell Tisslle Res 1993, 271 :33-45. review, have been highlighted as: Describes an electron microscopic method for detection of DNA of special interest in situ which combines high sensitivity with perfect ultrastructural 00 of outstanding interest preservation. The method is applicable to ultrathin sections of con­ ventionally fixed and embedded specimens. Free DNA ends that 1. Hadjiolov AA: 1be Nudeolus and Ribosome Biogenesis. are generated by the sectioning process are elongated by the en­ Wien: Springer Verlag; 1985. zyme terminal deoxynucleotidyl transferase in the presence of 5- 2. Reeder RH: rRNA Synthesis in the Nucieolus. Trends Genel bromodeoxyuridine 5'-triphosphate. Visualization ofthe tag is based 1990, 6:390-395. on immunogold electron microscopy. The authors present examples of the intranucleolar distribution of DNA in several species. 3. Paule MR: Polymerase I Transcription, Termination, and Pro­ cessing. Gene Expression 1993, 3:1-9. 20. Vandelaer M, Thiry M, Goessens G: Ultrastructural Distribu­ tion oC DNA within lhe Ring-Shaped Nucieolus oC Human 4. Olson MO): The Role oC Proteins in Nucleolar Structure Resling T Lymphocytes. Exp Gell Res 1993, 205:430-432. and Function. In 1be Eukaryolie NuciellS. Molecu/ar Bio­ 21. Thiry M: Uitrastructural Distribution of DNA and RNA within ebemis/ry and Macromolecu/ar Assemb/ies, Vol. 2. Edited the Nucieolus of Human Sertoli Cells as Seen by Molecular by Stmuss PR, Wilson SH. Caldwell: The Telford Press; Immunocytochemistry. J Gell Sci 1993, 105:33-39. 1990:519-559. 22. Tremblay SD, Gugg S, Lafontaine ]G: Ultrastructural, Cy­ 5. Sollner-Webb ß, Tyc K, Steitz ]: Ribosomal RNA Process­ tochemical and ImmunO<:ytochemical Investigation of the ing in Eukaryotcs. In Ribosoma/ RNA: S/nlcture, Evo/lIIion, Interphase Nucleus in the Unicellular Green Aiga Cblamy­ Processing and FunClion in Pro/ein Synrbesis. Edited by domonas reinbardtil. Biol Gell 1992, 76:73-86. Zimmerman R, Dahlberg A. ßoca Raton: CRC Press; 1994: in press. 23. Derenzini M, Farabegoli F, Trere D: Localization of DNA in the Fibrillar Components of the Nucleolus: a Cytochemi­ 6. Fournier M], Maxwell ES: The Nucieolar snRNAs: Catching cal and Morphomelric Study. J His/oebem Gy/ocbem 1993, up with the Spliceosomal snRNAs. Trends Biocbem Sei 1993, 41:829-836. 18:131-135. 24. Mosgöller W, Schöfer C, Derenzini M, Steiner M, Maier U, 7. Sollner-Webb ß: Novd Intron-Encoded Small Nucieolar Wachtier F: Distribution of DNA in Human Sertoli Cell Nu­ RNAs. Gell 1993, 75:403-405. c1eoli. J Histoebem Gylocbem 1993, 41:1487-1493. 8. Scheer U, ßenavente R: Functional and Dynamic Aspects of 25 . ]imenez-Garcfa LF, Segura-Valdez M, Ochs RL, Echeverria the Mammalian Nucieolus. BIoEssays 1990, 12:14-21. OM, Väzquez-Nin GH, ßusch H: Electron Microscopic Local­ 9. Warner ]R: The Nucieolus and Ribosome Formation. Gllrr ization of Ribosomal DNA in Rat Liver Nucleoli by Noniso­ Opin Gell Bio/I990, 2:521-527. topic in Situ Hybridization. Exp Gell Res 1993, 207:220-225. 26. Dundr M, Raska I: Nonisotopic Ultrastructural Mapping 10. Jordan EG: Interpreting Nucieolar Structure: Where Are the oe Transcription Sites within the Nucleolus. Exp Gell Res 1993, Transcribing Genes? J Gell Sei 1991 , 98:437-442. 208:275-281. 11. Fischer D, Weisenberger D, Scheer U: Assigning Functions Describes a non-isotopic method for the ultrastructural localization to Nucieolar Struclures. Gbromosoma 1991 , 101 :133-140. of RNA synthesis by means of 5-bromouridine 5'-triphosphate incor­ poration in perrneabilized cells. The transcription sites are mapped 12. Hernandez-Verdun D: The Nucieolus Today. J Gell Sci 1991, to the border region of fibrillar centers with the surrounding dense 99:465-471. fibrillar component. This method is superior to pulse-Iabeling experi­ 13. Scheer 11, Thiry M, Goessens G: Slructure, Function and As­ ments with radioactive precursors. As discussed by the authors, how­ sembly of the Nucieolus. Trends Gell Blo/I993, 3:236-241. ever, this method may lead to considerable structural distortions. 14 . Wachtier F, Stahl A: The Nucieolus: a Structural and Func­ 27 , Hozäk P, Cook PR, Schöfer C, Mosgöller W, Wachtier F: lional Interpretation. Mieron 1993, 24:473-505. Site of Transcription of Ribosomal RNA and Intranucleolar Structure in HeLa Cells. J Gell Sei 1994, 107:639-648. 0 15. Roussel P, Andre C, Massan C, Geraud G, Hernandez-Ver­ Uses an approach similar to that used in [26 ] with essentially the dun D: Localization of RNA Polymerase I Transcription Fac­ same results. A working model for nucleolar tmnscription is pre­ tor hUBF during the Cdl Cycie. J Gell Sei 1993, 104:327-337. sented. Active rRNA genes are attached through the polymerases to the surface of the fibrillar centers. 16. Zatsepina OV, Voit R, Grummt I, Spring H, Semenov MV, Trendelenburg MF: The RNA Polymerase I-Specific 28. Mougey Eß, O'Reilly M, Osheim Y, Miller OL ]r, ßeyer A, Transcription Initiation Factor UBF is Associated with Sollner-Webb B: The Terminal Balls Characteristic of Eukary­ Transcriptionally Active and Inactive Ribosomal Genes. otic rRNA Transcription Units in Chromatin Spreads are Gbromosoma 1993, 102:599-611. rRNA Processing Complexes. Genes Dev 1993, 7:1609-1619. The nucleolus Scheer and Weisenberger 359

In Miller spreads, the nascent transcripts of rRNA genes are charac­ 37. Peculis BA, Gall JG: Localization of the Nucleolar Protein terized by a conspicuous terminal thickening. Convincing evidence N038 in Amphibian Oocytes. J Gell Bio/l992, 116:1-14. is presented that these terminal balls are the structural counterparts 38. Wang D, Umekawa H, Olson MO]: Expression and Subcel­ of processing complexes that form at the 5' ends of growing pre­ lular Locations of Two Forms of Nucleolar Protein B23 in rRNAs. Several mutant rDNA constructs were injected into nuclei of Rat Tissues and Cells. Gell Mol Biol Res 1993, 39:33-42. XenopllS oocytes and their transcription units analyzed in electron microscopic spreads. An evolutionarily conserved sequence within 39. Maeda Y, Hisatake K, Kondo T, Hanada K, Song C-Z, the external transcribed spacer is shown to be required for terminal Nishimura T, Murdmatsu M: Mouse rRNA Gene Transcription ball formation. The same sequence is necessary for assembly of pro­ Factor mUBF Requires 80th HMG-8ox 1 and an Acidic Tail cessing complexes in biochemical experiments conducted in parallel for Nucleolar Accumulation: Molecular Analysis of the Nu­ [29--]. cleolar Targeting Mechanism. EMBO J 1992, 11 :3695-3704. 29. Mougey EB, Pape LK, Sollner-Webb 13: A U3 Sm all Nuclear 40. Trirnbur GM, Walsh CJ: Nucleolus-Like Morphology Pro- -- Ribonucleoprotein-Requiring Processing Event in the 5' Ex­ -- duced during the In Vltro Reassociation of Nudeolar ternal Transcribed Spacer of XenopllS Precursor rRNA. Mol Components. J Gell Biol 1993, 122:753-766. Gell Bio/l993, 13 :5990-5998. Salt extraction solubilizes ameba nucleoli; lowering the salt concen­ In this paper, the authors identify the processing site within the 5' tration enables them to reassociate. The in vitra assembled nucleoli external transcribed spacer of XenopltS. Downstream of this site resemble normal nucleoli in their size, protein composition and ul­ is a highly conserved RNA element that is required for assembly trastructure. This system should be useful for studying the factors of the processing complex. The processing complex is a relatively involved in nucleolus assembly and maintenance. large structure, with a sedimentation coefficient of 20S, consisting of 41. Karpen GH, Schaefer JE, Laird CD: A Drosophila rRNA Gene several proteins and U3 snoRNA. It is the morphological equivalent Located in Euchromatin is Active in Transcription and Nu­ to the terminal balls of the nascent pre-rRNAs [28--]. cleolus Formation. Genes Dev 1988, 2:1745-1763. 30. Schmidt-Zachmann MS , Nigg EA: Protein Localization to the 42. Azum-Gelade M-C, Noaillac-Depeyre J, Caizergues-Ferrer M, - Nucleolus: a Search for Targeting Domains in Nucleolin. J Gas N: Cell Cyde Redistribution of U3 snRNA and Fibril­ Gell Sei 1993, 105:799-806. larin. Presence in the Cytoplasmic Nucleolus Remnant and The authors have determined sequence requirements for nuclear im­ in the Prenucleolar 80dies at Telophase. J Gell Sei 1994, port and micleolar accumulation of the protein nucleolin. Nucleolar 107:463-475. targeting requires two types of RNA binding motifs in addition to the 43. Oakes M, Nogi Y, Clark MW, Nomura M: Structural A1ter- NLS . -- ations of the Nucleolus in Mutants of Saccharomyces cere­ 31. Messmer 13, Dreyer C: Requirements for Nuclear Transloca- vislae Defective in RNA Polymerase I. Mol Gell Biol 1993, - tion and Nucleolar Accumulation of Nucleolin of Xenopus 13:2441-2455. laevis. Eur J Gell Biol 1993, 61:369-382. A yeast mutant is described that lacks functional RNA polymerase I. During early development of Xenopus laevis, nuclear and nucleolar Ribosomal RNA is synthesized by polymerase II from a hybrid gene location of the protein nucleolin is temporally uncoupled. Nucleo­ .consisting of a pre-rRNA coding region fused to the GAL7 promotor. lar accumulation is shown to depend on two different RNA-binding In these mutants, the nucleolus is fragmented into mini-nucleolar motifs. The authors suggest that pre-rRNA may serve as a nucleolar bodies containing fibrillarin. The authors propose that active poly­ anchor for nucleolin. merase I may playa role as a structural element for the maintenance of nucleolar structure. A remarkable result is that the intact nucleolar 32. Creancier L, Prats H, Zanibellato C, Amalric F, Bugler 13: De- structure apparently is not required for ribosome biosynthesis. - termination of the Functional Domains Involved in Nucleolar Targeting of Nucleolin. Mol Biol Gell 1993, 4:1239-1250. 44. Yokoyama Y, Niwa K, Tamaya T: Scattering of the Silver­ Two RNA-binding domains of the protein nucleolin in combination Stained Proteins of Nucleolar Organizer Regions in Ishikawa with the NLS are sufficient to target a chloramphenicol acetyltrans­ Cells by Aetinomycin D. Exp Gell Res 1992, 202:77-86. ferase reporter gene to the nucleolus. 45. Bell P, Dabauvalle M-C, Scheer U: In Vltro Assembly of Prenudeolar 80dies in Xenopus Egg Extraet. J Gell Biol 33. Yan C, Melese T: Multiple Regions of NSRI Are Sufficient 1992, 118:1297-1304. for Accumulation of a Fusion Protein within the Nucleolus. J Gell Bio/l993, 123:1081-1091. 46. Wu Z, Murphy C, Wu C-HH, Tsvetkov A, GaU JG: Snurpo- The yeast nucleolar protein NSRI is structurally related to the mam­ - somes and Coiled Bodies. Gold Spring Harbor Symp Quant malian nucleolar protein nucleolin. Various regions of NSRI were Biol 1994, 58:in press. fused to areporter and the resulting hybrid proteins were tested for Structures resembling PNBs can be assembled In vitra using Xeno­ their ability to accumulate in the nucleolus. All three different re­ plIS egg extrac.t (see also (45)). The authors describe the presence gions of NSRI are Iikely to determine nucleolar accumulation. The of several spliceosomal snRNAs and the protein coilin in the 'syn­ authors emphasize that nucleolar targeting of different proteins does thetic' PNBs. A relationship between PNBs and coiled bodies is not involve a specific consensus sequence. emphasized. 34. Adachi Y, Copeland TD, Hatanaka M, Oroszlan S: Nucleolar 47. Lamond AI, Carmo-Fonseca M: The Coiled Body. Trends Targeting Signal of Rex Protein of Human T-Cell Leukemia Gell Biol 1993, 3:198-204. Virus Type I Specifically Binds to Nucleolar Shuttle Protein 48. Ochs RL, Stein TW Jr, Tan EM: Coiled Bodies in the Nude­ 6-23. J Biol Ghern 1993, 268:13930-13934. olus of Breast Cancer Cells. J Gell Sei 1994, 107:385-399. Rex protein, a regulatory factor of the retrovirus HTLV-I, is located in the nucleolus of infected cells, probably through specific interactions 49. Andrdde LEC, Tan EM, Chan EKL: Immunocytochemical with the nucleolar protein 1323. Analysis of the Coiled Body in the Cell Cyde and during Ceil Proliferation. Proe Natl Aead Sei USA 1993, 90:1947-1951. 35. Meier UT, Blobel G: Nopp140 Shuttles on Tracks between 50. Franke WW, Kleinschmidt JA, Spring H, Krohne G, Grund C, Nucleolus and Cytoplasm. Gell 1992, 70:127-138. Trendelenburg MF, Stoehr M, Scheer U: A Nucleolar Skeleton 36. Xue Z, Shan X, Lapeyre 13, Melese T: The Amino Termi- of Protein Filaments Demonstrated in Amplified Nucleoli of - nus of Mammalian Nucleolin Specifically Recognizes SV40 Xenopus laevis. J Gell Biol 1981, 90:289-299. T-Antigen Type Nuclear Localization Sequences. Eur J Gell Biol 1993, 62:13-21. Nucleolin belongs to the group of nucleolar proteins that shuttle be­ tween the nucleolus and the cytoplasm. Here it is shown that mam­ U Scheer, D Weisenberger, Department of Cell and Develop­ malian nucleolin contains an NLS recognition site. Involvement of mental Biology, Theodor-Boveri-Institute (Biocenter), University of nucleolin in nucleocytoplasmic transport processes is proposed. Würzburg, Am Hubland, D-97074 Würzburg, Germany.